Kunitz domain polypeptide Zkun11

ABSTRACT

Members of the serine protease family play a role in carefully controlled processes, such as blood coagulation, fibrinolysis, complement activation, fertilization, and hormone production. The enzymatic activity of the serine proteases is regulated in part by protease inhibitors that comprise a Kunitz domain. Zkun11 is a new member of the Kunitz domain protein family.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional application No.60/171,722 (filed Dec. 22, 1999), the contents of which are incorporatedby reference.

TECHNICAL FIELD

The present invention relates generally to a new gene that encodes anenzyme inhibitor. In particular, the present invention relates to anovel protein, designated “Zkun11,” and to nucleic acid moleculesencoding Zkun11.

BACKGROUND OF THE INVENTION

In animals, proteinases are important in wound healing, extracellularmatrix destruction, tissue reorganization, and in cascades leading toblood coagulation, fibrinolysis, and complement activation. Proteinasesare released by inflammatory cells for destruction of pathogens orforeign materials, and by normal and cancerous cells as they movethrough their surroundings.

The activity of proteinases is regulated by inhibitors (Roberts et al.,Critical Reviews in Eukaryotic Gene Expression 5:385 (1995)). One familyof proteinase inhibitors, the Kunitz inhibitors, includes inhibitors oftrypsin, chymotrypsin, elastase, kallikrein, plasmin, coagulationfactors XIa and IXa, and cathepsin G. These inhibitors thus regulate avariety of physiological processes, including blood coagulation,fibrinolysis, and inflammation.

Proteinase inhibitors regulate the proteolytic activity of targetproteinases by occupying the active site and thereby preventingoccupation by normal substrates. Although proteinase inhibitors fallinto several unrelated structural classes, they all possess an exposedloop (variously termed an “inhibitor loop,” a “reactive core,” a“reactive site,” or a “binding loop”), which is stabilized byintermolecular interactions between residues flanking the loop and theprotein core (Bode and Huber, Eur. J. Biochem. 204:433 (1992)).Interaction between inhibitor and enzyme produces a stable complex,which disassociates very slowly, releasing either uncleaved inhibitor,or a modified inhibitor that is cleaved at the scissile bond of thebinding loop.

One class of proteinase inhibitors, the Kunitz inhibitors, are generallybasic, low molecular weight proteins comprising one or more inhibitorydomains (“Kunitz domains”). The Kunitz domain is a folding domain ofapproximately 50-60 residues, which forms a central anti-parallel betasheet and a short C-terminal helix. This characteristic domain comprisessix cysteine residues that form three disulfide bonds, resulting in adouble-loop structure. Between the N-terminal region and the first betastrand resides the active inhibitory binding loop. This binding loop isdisulfide bonded through the P2 Cys residue to the hairpin loop formedbetween the last two beta strands. Isolated Kunitz domains from avariety of proteinase inhibitors have been shown to have inhibitoryactivity (see, for example, Petersen et al., Eur. J. Biochem. 125:310(1996); Wagner et al., Biochem. Biophys. Res. Comm. 186:1138 (1992);Dennis et al., J. Biol. Chem. 270:25411 (1995)).

Proteinase inhibitors comprising one or more Kunitz domains includetissue factor pathway inhibitor (TFPI), tissue factor pathway inhibitor2 (TFPI-2), amyloid β-protein precursor (AβPP), aprotinin, and placentalbikunin. TFPI, an extrinsic pathway inhibitor and a naturalanticoagulant, contains three tandemly linked Kunitz inhibitor domains.The amino-terminal Kunitz domain inhibits factor VIIa, plasmin, andcathepsin G; the second domain inhibits factor Xa, trypsin, andchymotrypsin; and the third domain has no known activity (Petersen etal., Eur. J. Biochem. 125:310 (1996)). TFPI-2 has been shown to be aninhibitor of the amidolytic and proteolytic activities of human factorVIIa-tissue factor complex, factor XIa, plasma kallikrein, and plasmin(Sprecher et al., Proc. Nat'l Acad. Sci. USA 91:3353 (1994); Petersen etal., Biochem. 35:266 (1996)). The ability of TFPI-2 to inhibit thefactor VIIa-tissue factor complex and its relatively high levels oftranscription in umbilical vein endothelial cells, placenta and liversuggests a specialized role for this protein in hemostasis (Sprecher etal., Proc. Nat'l Acad. Sci. USA 91:3353 (1994)).

Aprotinin (bovine pancreatic trypsin inhibitor) is a broad spectrumKunitz-type serine proteinase inhibitor that has been shown to preventactivation of the clotting cascade. Aprotinin is a moderate inhibitor ofplasma kallikrein and plamin, and blockage of fibrinolysis andextracorporeal coagulation have been detected in patients givenaprotinin during open heart surgery (Davis and Whittington, Drugs 49:954(1995); Dietrich et al., Thorac. Cardiovasc. Surg. 37:92 (1989)).Aprotinin has also been used in the treatment of septic shock, adultrespiratory distress syndrome, acute pancreatitis, hemorrhagic shock,and other conditions (Westaby, Ann. Thorac. Surg. 55:1033 (1993);Wachtfogel et al., J. Thorac. Cardiovasc. Surg. 106:1 (1993)). Theclinical utility of aprotinin is believed to arise from its inhibitoryactivity towards plasma kallikrein or plasmin (Dennis et al., J. Biol.Chem. 270:25411 (1995)). Placental bikunin is a serine proteinaseinhibitor containing two Kunitz domains (Delaria et al., J. Biol. Chem.272:12209 (1997)). Individual Kunitz domains of bikunin have beenexpressed and shown to be potent inhibitors of trypsin, chymotrypsin,plasmin, factor XIa, and tissue and plasma kallikrein (Delaria et al.,J. Biol. Chem. 272:12209 (1997)).

Known Kunitz-type inhibitors lack specificity and may have low potency.Lack of specificity can result in undesirable side effects, such asnephrotoxicity that occurs after repeated injections of high doses ofaprotinin. These limitations may be overcome by preparing isolatedKunitz domains, which may have fewer side effects than traditionalanticoagulants. Hence, there is a need in the art for additionalKunitz-type proteinase inhibitors.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a novel Kunitz inhibitor protein,designated “Zkun11.” The present invention also provides Zkun11 variantpolypeptides and Zkun11 fusion proteins, as well as nucleic acidmolecules encoding such polypeptides and proteins, and methods for usingthese nucleic acid molecules and amino acid sequences.

DESCRIPTION OF THE INVENTION

1. Overview

The present invention provides nucleic acid molecules that encode a newhuman Kunitz inhibitor protein, designated as “Zkun11.” An illustrativenucleotide sequence that encodes Zkun11 is provided by SEQ ID NO:1. Theencoded polypeptide has the following amino acid sequence: CNYPLKKGTCNSYLTRFYYN TLTFLCEPFV FSGCGGNRNN FKQKYFCEKM C (SEQ ID NO:2).

Sequence analysis revealed that Zkun11 can be represented by thefollowing Kunitz domain sequence: C-X(8)-C-X(15)-C-X(7)-C-X(12)-C-X(3)-C(SEQ ID NO:5), where “X” is any amino acid residue and the values inparentheses indicate the number of such variable residues. Referring toSEQ ID NO:2, disulfide bonds are predicted to be formed by pairedcysteine residues Cys1 to Cys51, Cys10 to Cys34, and Cys26 to Cys47.Amino acid substitutions can be made within the Zkun11 sequence so longas the conserved cysteine residues are retained and the higher orderstructure is not disrupted.

Example 1 illustrates that the Zkun11 gene is expressed differentially.For example, the Zkun11 gene is expressed in prostate tissue, but is notexpressed in a variety of tissues including adrenal gland, bladder,colon, kidney, liver, ovary, pituitary, placenta, rectum, spleen,stomach, and testis. Thus, prostate tissue can be distinguished fromother tissues identified herein, using nucleic acid molecules thatencode Zkun11, as well as Zkun11 polypeptides, and antibodies (orantibody fragments) to Zkun11 polypeptides.

As detailed below, the present invention provides isolated polypeptideshaving an amino acid sequence that is at least 70%, at least 80%, or atleast 90% identical to the amino acid sequence of SEQ ID NO:2, whereinsuch isolated polypeptides specifically bind with an antibody thatspecifically binds with a polypeptide having the amino acid sequence ofSEQ ID NO:2. Typically, such isolated polypeptides comprise an aminoacid sequence with the structure: C-X(8)-C-X(15)-C-X(7)-C-X(12)-C-X(3)-C (SEQ ID NO:5), where “X” is any amino acid residueand the values in parentheses indicate the number of such variableresidues. An illustrative polypeptide is a polypeptide that comprisesthe amino acid sequence of SEQ ID NO:2.

The present invention also provides isolated polypeptides, comprising aKunitz domain amino acid sequence, wherein the Kunitz domain amino acidsequence shares a percent identity with the amino acid sequence of SEQID NO:2, wherein the percent identity is selected from the groupconsisting of 70%, 80%, 85%, and at least 90%, and wherein the isolatedpolypeptide comprises an amino acid sequence with the structure:C-X(8)-C-X(15)-C-X(7)-C-X(12)-C-X(3)-C (SEQ ID NO:5), where “X” is anyamino acid residue and the values in parentheses indicate the number ofsuch variable residues. Isolated polypeptides can further comprise asecond Kunitz domain amino acid sequence, wherein the first and secondKunitz domain sequences are separated by a linker that comprises atleast about ten amino acid residues. Such linkers can comprise, forexample, about 20, about 40, about 60, or about 80, amino acid residues.Additional Kunitz domain inhibitors can be added in a similar manner.

Particular polypeptides of the present invention can also becharacterized by serine protease inhibitory activity. Moreover, peptidesand polypeptides of the present invention can further comprise anaffinity tag.

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude polyclonal antibodies, murine monoclonal antibodies, humanizedantibodies derived from murine monoclonal antibodies, and humanmonoclonal antibodies. Illustrative antibody fragments include F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, and minimal recognition units. The presentinvention also includes anti-idiotype antibodies that bind with suchantibodies and antibody fragments, as well as compositions comprising acarrier and a peptide, polypeptide, antibody, or anti-idiotype antibodydescribed herein.

The present invention also provides isolated nucleic acid molecules thatencode a Zkun11 polypeptide, wherein the nucleic acid molecule isselected from the group consisting of: a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:3; a nucleic acidmolecule encoding the amino acid sequence of SEQ ID NO:2; and a nucleicacid molecule that remains hybridized following stringent washconditions to a nucleic acid molecule consisting of the nucleotidesequence of SEQ ID NO:1, or to the complement of the nucleotide sequenceof SEQ ID NO:1.

Illustrative nucleic acid molecules include those in which anydifference between the amino acid sequence encoded by the nucleic acidmolecule and the corresponding amino acid sequence of SEQ ID NO:2 is dueto a conservative amino acid substitution. The present invention furthercontemplates isolated nucleic acid molecules that comprise, or consistof, the nucleotide sequence of SEQ ID NO:1.

The present invention also includes vectors and expression vectorscomprising such nucleic acid molecules. Such expression vectors maycomprise a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator. The present invention further includesrecombinant host cells comprising these vectors and expression vectors.Illustrative host cells include bacterial, yeast, fungal, avian, insect,mammalian, and plant cells. Recombinant host cells comprising suchexpression vectors can be used to produce Zkun11 polypeptides byculturing such recombinant host cells that comprise the expressionvector and that produce the Zkun11 protein, and, optionally, isolatingthe Zkun11 protein from the cultured recombinant host cells. The presentinvention further includes products made by such processes.

The present invention also contemplates methods for detecting thepresence of Zkun11 RNA in a biological sample, comprising the steps of(a) contacting a Zkun11 nucleic acid probe under hybridizing conditionswith either (i) test RNA molecules isolated from the biological sample,or (ii) nucleic acid molecules synthesized from the isolated RNAmolecules, wherein the probe has a nucleotide sequence comprising aportion of the nucleotide sequence of SEQ ID NO:1, or its complement,and (b) detecting the formation of hybrids of the nucleic acid probe andeither the test RNA molecules or the synthesized nucleic acid molecules,wherein the presence of the hybrids indicates the presence of Zkun11 RNAin the biological sample. An example of a biological sample is a humanbiological sample, such as a biopsy or autopsy specimen.

The present invention further provides methods for detecting thepresence of Zkun11 polypeptide in a biological sample, comprising thesteps of: (a) contacting the biological sample with an antibody or anantibody fragment that specifically binds with a polypeptide having theamino acid sequence of SEQ ID NO:2, wherein the contacting is performedunder conditions that allow the binding of the antibody or antibodyfragment to the biological sample, and (b) detecting any of the boundantibody or bound antibody fragment. Such an antibody or antibodyfragment may further comprise a detectable label selected from the groupconsisting of radioisotope, fluorescent label, chemiluminescent label,enzyme label, bioluminescent label, and colloidal gold. An example of abiological sample is a human biological sample, such as a biopsy orautopsy specimen.

The present invention also provides kits for performing these detectionmethods. For example, a kit for detection of Zkun11 gene expression maycomprise a container that comprises a nucleic acid molecule, wherein thenucleic acid molecule is selected from the group consisting of (a) anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,(b) a nucleic acid molecule comprising the complement of the nucleotidesequence of SEQ ID NO:1, (c) a nucleic acid molecule that is a fragmentof (a) consisting of at least eight nucleotides, and (d) a nucleic acidmolecule that is a fragment of (b) consisting of at least eightnucleotides. Such a kit may also comprise a second container thatcomprises one or more reagents capable of indicating the presence of thenucleic acid molecule. On the other hand, a kit for detection of Zkun11protein may comprise a container that comprises an antibody, or anantibody fragment, that specifically binds with a polypeptide having theamino acid sequence of SEQ ID NO:2.

The present invention also provides fusion proteins, comprising a Zkun11polypeptide moiety. Such fusion proteins can further comprise animmunoglobulin moiety. In such fusion proteins, the immunoglobulinmoiety may be an immunoglobulin heavy chain constant region, such as ahuman F_(c) fragment. The present invention further includes isolatednucleic acid molecules that encode such fusion proteins.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SPI, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner. For example, theZkun11 regulatory element preferentially induces gene expression inspleen, thymus, spinal cord, and lymph node tissues, as opposed toplacenta, lung, and liver tissues.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, which has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces Zkun11from an expression vector. In contrast, Zkun11 can be produced by a cellthat is a “natural source” of Zkun11, and that lacks an expressionvector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a Zkun11polypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of Zkun11using affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a nucleotide sequence thatencodes a peptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger polypeptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of an anti-Zkun11antibody, and thus, an anti-idiotype antibody mimics an epitope ofZkun11. Particular Zkun11 anti-idiotype antibodies possess serineprotease inhibitor activity.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-Zkun11 monoclonal antibody fragment bindswith an epitope of Zkun11.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, a “therapeutic agent” is a molecule or atom, which isconjugated to an antibody moiety to produce a conjugate, which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom, which can be conjugated toan antibody moiety to produce a molecule useful for diagnosis. Examplesof detectable labels include chelators, photoactive agents,radioisotopes, fluorescent agents, paramagnetic ions, or other markermoieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu—Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). Nucleic acid molecules encoding affinity tagsare available from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for Zkun11” or an “Zkun11anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the Zkun11 gene,or (b) capable of forming a stable duplex with a portion of an mRNAtranscript of the Zkun11 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant Zkun11 gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:2. Such variants include naturally-occurringpolymorphisms of Zkun11 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:2. Additional variant forms of Zkun11 genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant Zkun11 gene can be identified bydetermining whether the gene hybridizes with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1, or its complement, understringent conditions.

Alternatively, variant Zkun11 genes can be identified by sequencecomparison. Two amino acid sequences have “100% amino acid sequenceidentity” if the amino acid residues of the two amino acid sequences arethe same when aligned for maximal correspondence. Similarly, twonucleotide sequences have “100% nucleotide sequence identity” if thenucleotide residues of the two nucleotide sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art (see, for example, Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify a variant Zkun11gene or variant Zkun11 polypeptide, a variant gene or polypeptideencoded by a variant gene may be characterized by at least one of: theability to bind specifically to an anti-Zkun11 antibody, and serineprotease inhibitor activity.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of Zkun11 genes.Within the context of this invention, a “functional fragment” of aZkun11 gene refers to a nucleic acid molecule that encodes a portion ofa Zkun11 polypeptide, which specifically binds with an anti-Zkun11antibody or possesses serine protease inhibitor activity. For example, afunctional fragment of a Zkun11 gene described herein comprises aportion of the nucleotide sequence of SEQ ID NO:1, and encodes apolypeptide that specifically binds with an anti-Zkun11 antibody.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

3. Production of Nucleic Acid Molecules that Encode Zkun11

Nucleic acid molecules encoding a human Zkun11 gene can be obtained byscreening a human cDNA or genomic library using polynucleotide probesbased upon SEQ ID NO:1. These techniques are standard andwell-established.

As an illustration, a nucleic acid molecule that encodes a human Zkun11gene can be isolated from a human cDNA library. In this case, the firststep would be to prepare the cDNA library using methods well-known tothose of skill in the art. In general, RNA isolation techniques mustprovide a method for breaking cells, a means of inhibitingRNase-directed degradation of RNA, and a method of separating RNA fromDNA, protein, and polysaccharide contaminants. For example, total RNAcan be isolated by freezing tissue in liquid nitrogen, grinding thefrozen tissue with a mortar and pestle to lyse the cells, extracting theground tissue with a solution of phenol/chloroform to remove proteins,and separating RNA from the remaining impurities by selectiveprecipitation with lithium chloride (see, for example, Ausubel et al.(eds.), Short Protocols in Molecular Biology, 3^(rd) Edition, pages 4-1to 4-6 (John Wiley & Sons 1995) [“Ausubel (1995)”]; Wu et al., Methodsin Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) [“Wu(1997)”]). Alternatively, total RNA can be by extracting ground tissuewith guanidinium isothiocyanate, extracting with organic solvents, andseparating RNA from contaminants using differential centrifugation (see,for example, Chirgwin et al., Biochemistry 18:52 (1979); Ausubel (1995)at pages 4-1 to 4-6; Wu (1997 ) at pages 33 -41).

In order to construct a cDNA library, poly(A)⁺ RNA must be isolated froma total RNA preparation. Poly(A)⁺ RNA can be isolated from total RNAusing the standard technique of oligo(dT)-cellulose chromatography (see,for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972);Ausubel (1995) at pages 4-11 to 4 -12).

Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNA usingtechniques well-known to those in the art. (see, for example, Wu (1997)at pages 41-46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules. For example, such kits areavailable from Life Technologies, Inc. (Gaithersburg, Md.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Promega Corporation (Madison,Wis.) and STRATAGENE (La Jolla, Calif.).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector. See, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λgt11, ” inDNA Cloning: A Practical Approach Vol. 1, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47-52.

Alternatively, double-stranded cDNA molecules can be inserted into aplasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla,Calif.), a LAMDAGEM-4 (Promega Corp.) or other commercially availablevectors. Suitable cloning vectors also can be obtained from the AmericanType Culture Collection (Manassas, Va.).

To amplify the cloned cDNA molecules, the cDNA library is inserted intoa prokaryotic host, using standard techniques. For example, a cDNAlibrary can be introduced into competent E. coli DH5 cells, which can beobtained, for example, from Life Technologies, Inc. (Gaithersburg, Md.).

A human genomic library can be prepared by means well-known in the art(see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well-known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307 -327).

Nucleic acid molecules that encode a human Zkun11 gene can also beobtained using the polymerase chain reaction (PCR) with oligonucleotideprimers having nucleotide sequences that are based upon the nucleotidesequences of the human Zkun11 gene, as described herein. General methodsfor screening libraries with PCR are provided by, for example, Yu etal., “Use of the Polymerase Chain Reaction to Screen Phage Libraries,”in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).Moreover, techniques for using PCR to isolate related genes aredescribed by, for example, Preston, “Use of Degenerate OligonucleotidePrimers and the Polymerase Chain Reaction to Clone Gene Family Members,”in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White (ed.), pages 317 -337 (Humana Press, Inc. 1993).

Alternatively, human genomic libraries can be obtained from commercialsources such as Research Genetics (Huntsville, Ala.) and the AmericanType Culture Collection (Manassas, Va.).

A library containing cDNA or genomic clones can be screened with one ormore polynucleotide probes based upon SEQ ID NO:1, using standardmethods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).

Anti-Zkun11 antibodies, produced as described below, can also be used toisolate DNA sequences that encode human Zkun11 genes from cDNAlibraries. For example, the antibodies can be used to screen λgt11expression libraries, or the antibodies can be used for immunoscreeningfollowing hybrid selection and translation (see, for example, Ausubel(1995) at pages 6-12 to 6-16; Margolis et al., “Screening λ expressionlibraries with antibody and protein probes,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14(Oxford University Press 1995)).

As an alternative, a Zkun11 gene can be obtained by synthesizing nucleicacid molecules using mutually priming long oligonucleotides and thenucleotide sequences described herein (see, for example, Ausubel (1995)at pages 8-8 to 8-9). Established techniques using the polymerase chainreaction provide the ability to synthesize DNA molecules at least twokilobases in length (Adang et al., Plant Molec. Biol. 21:1131 (1993),Bambot et al., PCR Methods and Applications 2:266 (1993), Dillon et al.,“Use of the Polymerase Chain Reaction for the Rapid Construction ofSynthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263-268,(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.4:299 (1995)).

The nucleic acid molecules of the present invention can also besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

The sequence of a Zkun11 cDNA or Zkun11 genomic fragment can bedetermined using standard methods. Zkun11 polynucleotide sequencesdisclosed herein can also be used as probes or primers to clone 5′non-coding regions of a Zkun11 gene. Promoter elements from a Zkun11gene can be used to direct the expression of heterologous genes in, forexample, transgenic animals or patients undergoing gene therapy. Theidentification of genomic fragments containing a Zkun11 promoter orregulatory element can be achieved using well-established techniques,such as deletion analysis (see, generally, Ausubel (1995)).

Cloning of 5′ flanking sequences also facilitates production of Zkun11proteins by “gene activation,” as disclosed in U.S. Pat. No. 5,641,670.Briefly, expression of an endogenous Zkun11 gene in a cell is altered byintroducing into the Zkun11 locus a DNA construct comprising at least atargeting sequence, a regulatory sequence, an exon, and an unpairedsplice donor site. The targeting sequence is a Zkun11 5′ non-codingsequence that permits homologous recombination of the construct with theendogenous Zkun11 locus, whereby the sequences within the constructbecome operably linked with the endogenous Zkun11 coding sequence. Inthis way, an endogenous Zkun11 promoter can be replaced or supplementedwith other regulatory sequences to provide enhanced, tissue-specific, orotherwise regulated expression.

4. Production of Zkun11 Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules that encode the Zkun11 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:3is a degenerate nucleotide sequence that encompasses all nucleic acidmolecules that encode the Zkun11 polypeptide of SEQ ID NO:2. Thoseskilled in the art will recognize that the degenerate sequence of SEQ IDNO:3 also provides all RNA sequences encoding SEQ ID NO:2, bysubstituting U for T. Thus, the present invention contemplates Zkun11polypeptide-encoding nucleic acid molecules comprising nucleotides 1 to153 of SEQ ID NO:1, and their RNA equivalents.

Table 1 sets forth the one-letter codes used within SEQ ID NO:3 todenote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:3, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

TABLE 2 Amino One Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NOs:2 and 3. Variant sequences can be readily testedfor functionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al., Nuc. Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. biotechnol. 6:494 (1995),and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (see Table 2). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequence disclosed in SEQ ID NO:3 serves as a templatefor optimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein. Sequencescontaining preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

The present invention further provides variant polypeptides and nucleicacid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are Zkun11 polypeptidesfrom other mammalian species, including porcine, ovine, bovine, canine,feline, equine, and other primate polypeptides. Orthologs of humanZkun11 can be cloned using information and compositions provided by thepresent invention in combination with conventional cloning techniques.For example, a cDNA can be cloned using mRNA obtained from a tissue orcell type that expresses Zkun11 as disclosed herein. Suitable sources ofmRNA can be identified by probing northern blots with probes designedfrom the sequences disclosed herein. A library is then prepared frommRNA of a positive tissue or cell line.

A Zkun11-encoding cDNA can then be isolated by a variety of methods,such as by probing with a complete or partial human cDNA or with one ormore sets of degenerate probes based on the disclosed sequences. A cDNAcan also be cloned using the polymerase chain reaction with primersdesigned from the representative human Zkun11 sequences disclosedherein. Within an additional method, the cDNA library can be used totransform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to Zkun11 polypeptide. Similartechniques can also be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human Zkun11, and that allelicvariation and alternative splicing are expected to occur. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the nucleotide sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:2. cDNA molecules generated from alternatively spliced mRNAs, whichretain the properties of the Zkun11 polypeptide are included within thescope of the present invention, as are polypeptides encoded by suchcDNAs and mRNAs. Allelic variants and splice variants of these sequencescan be cloned by probing cDNA or genomic libraries from differentindividuals or tissues according to standard procedures known in theart.

Within certain embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules comprising nucleotide sequences disclosed herein. For example,such nucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules consisting of the nucleotide sequence of SEQ IDNO:1, or to nucleic acid molecules consisting of a nucleotide sequencecomplementary to SEQ ID NO:1. In general, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polypeptide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions which influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5 -10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contribute positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazz-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration ofthe buffer, the stability of the hybrid is increased. Typically,hybridization buffers contain from between 10 mM-1 M Na⁺. The additionof destabilizing or denaturing agents such as formamide,tetralkylammonium salts, guanidinium cations or thiocyanate cations tothe hybridization solution will alter the T_(m) of a hybrid. Typically,formamide is used at a concentration of up to 50% to allow incubationsto be carried out at more convenient and lower temperatures. Formamidealso acts to reduce non-specific background when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant Zkun11polypeptide can be hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) at 42° C.overnight in a solution comprising 50% formamide, 5×SSC (1×SSC: 0.15 Msodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution (100×Denhardt's solution: 2% (w/v) Ficoll400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin,10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.One of skill in the art can devise variations of these hybridizationconditions. For example, the hybridization mixture can be incubated at ahigher temperature, such as about 65° C., in a solution that does notcontain formamide. Moreover, premixed hybridization solutions areavailable (e.g., EXPRESSHYB Hybridization Solution from CLONTECHLaboratories, Inc.), and hybridization can be performed according to themanufacturer's instructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. As an illustration, certain nucleicacid molecules encoding a variant Zkun11 polypeptide remain hybridizedfollowing stringent washing conditions with a nucleic acid moleculeconsisting of the nucleotide sequence of SEQ ID NO:1 (or itscomplement), in which the wash stringency is equivalent to 0.5×-2×SSCwith 0.1% SDS at 55-65° C., including 0.5×SSC with 0.1% SDS at 55° C.,or 2×SSC with 0.1% SDS at 65° C. One of skill in the art can readilydevise equivalent conditions, for example, by substituting the SSPE forSSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.5×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. For example, certain nucleic acid molecules encoding a variantZkun11 polypeptide remain hybridized following stringent washingconditions with a nucleic acid molecule consisting of the nucleotidesequence of SEQ ID NO:1 (or its complement), in which the washstringency is equivalent to 0.5×-0.2×SSC with 0.1% SDS at 50-65° C.,including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at65° C.

The present invention also provides isolated Zkun11 polypeptides thathave a substantially similar sequence identity to the polypeptide of SEQID NO:2, or orthologs. The term “substantially similar sequenceidentity” is used herein to denote polypeptides having 70%, 80%, 90%,95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown inSEQ ID NO:2.

The present invention also contemplates Zkun11 variant nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NO:2, and a hybridization assay, as described above.Such Zkun11 variants include nucleic acid molecules (1) that remainhybridized following stringent washing conditions with a nucleic acidmolecule consisting of the nucleotide sequence of SEQ ID NO:1 (or itscomplement), in which the wash stringency is equivalent to 0.5×-2×SSCwith 0.1% SDS at 55-65° C., and (2) that encode a polypeptide having70%, 80%, 90 %, 95% 96%, 97%, 98% or 99% sequence identity to the aminoacid sequence of SEQ ID NO:2.

Alternatively, Zkun11 variants can be characterized as nucleic acidmolecules (1) that remain hybridized following highly stringent washingconditions with a nucleic acid molecule consisting of the nucleotidesequence of SEQ ID NO:1 (or its complement), in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and(2) that encode a polypeptide having 70%, 80%, 90%, 95%, 96%, 97%, 98%or 99% sequence identity to the amino acid sequence of SEQ ID NO:2.

The present invention also includes Zkun11 variants that possess serineprotease inhibitor activity. Moreover, particular Zkun11 variants arecharacterized using hybridization analysis with a reference nucleic acidmolecule that is a fragment of a nucleic acid molecule consisting of thenucleotide sequence of SEQ ID NO:1, or its complement.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100).

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativeZkun11 variant. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequencesimilarity by identifying regions shared by the query sequence (e.g.,SEQ ID NO:2) and a test sequence that have either the highest density ofidentities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdescribed above.

The present invention includes nucleic acid molecules that encode apolypeptide having a conservative amino acid change, compared with theamino acid sequence of SEQ ID NO:2. That is, variants can be obtainedthat contain one or more amino acid substitutions of SEQ ID NO:2, inwhich an alkyl amino acid is substituted for an alkyl amino acid in aZkun11 amino acid sequence, an aromatic amino acid is substituted for anaromatic amino acid in a Zkun11 amino acid sequence, a sulfur-containingamino acid is substituted for a sulfur-containing amino acid in a Zkun11amino acid sequence, a hydroxy-containing amino acid is substituted fora hydroxy-containing amino acid in a Zkun11 amino acid sequence, anacidic amino acid is substituted for an acidic amino acid in a Zkun11amino acid sequence, a basic amino acid is substituted for a basic aminoacid in a Zkun11 amino acid sequence, or a dibasic monocarboxylic aminoacid is substituted for a dibasic monocarboxylic amino acid in a Zkun11amino acid sequence.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine.

The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

Particular variants of Zkun11 are characterized by having greater than96%, at least 97%, at least 98%, or at least 99% sequence identity tothe corresponding amino acid sequence (e.g., SEQ ID NO:2), wherein thevariation in amino acid sequence is due to one or more conservativeamino acid substitutions.

Conservative amino acid changes in a Zkun11 gene can be introduced bysubstituting nucleotides for the nucleotides recited in SEQ ID NO:1.Such “conservative amino acid” variants can be obtained, for example, byoligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like (seeAusubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), DirectedMutagenesis: A Practical Approach (IRL Press 1991)).

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4 -hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4 -azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for Zkun11 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., J.Biol. Chem. 271:4699 (1996). The identities of essential amino acids canalso be inferred from analysis of homologies with other serine proteaseinhibitors.

The location of Zkun11 activity subdomains can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306(1992), Smith et al., J. Mol. Biol. 224:899 (1992), and Wlodaver et al.,FEBS Lett. 309:59 (1992). Moreover, Zkun11 labeled with biotin or FITCcan be used for expression cloning of Zkun11 substrates and inhibitors.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

Variants of the disclosed Zkun11 nucleotide and polypeptide sequencescan also be generated through DNA shuffling as disclosed by Stemmer,Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747(1994), and international publication No. WO 97/20078. Briefly, variantDNAs are generated by in vitro homologous recombination by randomfragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNAs, such as allelic variants orDNAs from different species, to introduce additional variability intothe process. Selection or screening for the desired activity, followedby additional iterations of mutagenesis and assay provides for rapid“evolution” of sequences by selecting for desirable mutations whilesimultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-Zkun11 antibodies, can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The present invention also includes “functional fragments” of Zkun11polypeptides and nucleic acid molecules encoding such functionalfragments. Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a Zkun11 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. One alternative toexonuclease digestion is to use oligonucleotide-directed mutagenesis tointroduce deletions or stop codons to specify production of a desiredfragment. Alternatively, particular fragments of a Zkun11 gene can besynthesized using the polymerase chain reaction.

As an illustration, studies on the truncation at either or both terminiof interferons have been summarized by Horisberger and Di Marco,Pharmac. Ther. 66:507 (1995). Moreover, standard techniques forfunctional analysis of proteins are described by, for example, Treuteret al., Molec. Gen. Genet. 240:113 (1993), Content et al., “Expressionand preliminary deletion analysis of the 42 kDa 2-5A synthetase inducedby human interferon,” in Biological Interferon Systems, Proceedings ofISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72(Nijhoff 1987), Herschman, “The EGF Receptor,” in Control of Animal CellProliferation, Vol. 1, Boynton et al., (eds.) pages 169-199 (AcademicPress 1985), Coumailleau et al., J. Biol. Chem. 270:29270 (1995);Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al.,Biochem. Pharmacol. 50:1295 (1995), and Meisel et al., Plant Molec.Biol. 30:1 (1996).

The present invention also contemplates functional fragments of a Zkun11gene that has amino acid changes, compared with the amino acid sequenceof SEQ ID NO:2. A variant Zkun11 gene can be identified on the basis ofstructure by determining the level of identity with nucleotide and aminoacid sequences of SEQ ID NOs:1 and 2, as discussed above. An alternativeapproach to identifying a variant gene on the basis of structure is todetermine whether a nucleic acid molecule encoding a potential variantZkun11 gene can hybridize to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1, as discussed above.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a Zkun11 polypeptide describedherein. Such fragments or peptides may comprise an “immunogenicepitope,” which is a part of a protein that elicits an antibody responsewhen the entire protein is used as an immunogen. Immunogenicepitope-bearing peptides can be identified using standard methods (see,for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Antibodies that recognize short linear epitopes areparticularly useful in analytic and diagnostic applications that usedenatured protein, such as Western analysis, or in the analysis of fixedcells or tissue samples. Antibodies to linear epitopes are also usefulfor detecting fragments of Zkun11, such as might occur in body fluids orculture media. Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides can contain at leastfour to ten amino acids, at least ten to fifteen amino acids, or about15 to about 30 amino acids of SEQ ID NO:2. Such epitope-bearing peptidesand polypeptides can be produced by fragmenting a Zkun11 polypeptide, orby chemical peptide synthesis, as described herein. Moreover, epitopescan be selected by phage display of random peptide libraries (see, forexample, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), andCortese et al., Curr. Opin. Biotechnol. 7:616 (1996)). Standard methodsfor identifying epitopes and producing antibodies from small peptidesthat comprise an epitope are described, for example, by Mole, “EpitopeMapping,” in Methods in Molecular Biology, Vol. 10, Manson (ed.), pages105-116 (The Humana Press, Inc. 1992), Price, “Production andCharacterization of Synthetic Peptide-Derived Antibodies,” in MonoclonalAntibodies: Production, Engineering, and Clinical Application, Ritterand Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), andColigan et al. (eds.), Current Protocols in Immunology, pages9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons 1997).

For any Zkun11 polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise Zkun11 variants based upon thenucleotide and amino acid sequences described herein. Accordingly, thepresent invention includes a computer-readable medium encoded with adata structure that provides at least one of SEQ ID NO:1, SEQ ID NO:2,and SEQ ID NO:3. Suitable forms of computer-readable media includemagnetic media and optically-readable media. Examples of magnetic mediainclude a hard or fixed drive, a random access memory (RAM) chip, afloppy disk, digital linear tape (DLT), a disk cache, and a ZIP disk.Optically readable media are exemplified by compact discs (e.g., CD-readonly memory (ROM), CD-rewritable (RW), and CD-recordable), and digitalversatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

5. Production of Zkun11 Fusion Proteins

Fusion proteins of Zkun11 can be used to express Zkun11 in a recombinanthost, and to isolate expressed Zkun11. One type of fusion proteincomprises a peptide that guides a Zkun11 polypeptide from a recombinanthost cell. To direct a Zkun11 polypeptide into the secretory pathway ofa eukaryotic host cell, a secretory signal sequence (also known as asignal peptide, a leader sequence, prepro sequence or pre sequence) isprovided in the Zkun11 expression vector. While the secretory signalsequence may be derived from Zkun11, a suitable signal sequence may alsobe derived from another secreted protein or synthesized de novo. Thesecretory signal sequence is operably linked to a Zkun11-encodingsequence such that the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the nucleotide sequence encoding thepolypeptide of interest, although certain secretory signal sequences maybe positioned elsewhere in the nucleotide sequence of interest (see,e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat.No. 5,143,830).

While the secretory signal sequence of Zkun11 or another proteinproduced by mammalian cells (e.g., tissue-type plasminogen activatorsignal sequence, as described, for example, in U.S. Pat. No. 5,641,655)is useful for expression of Zkun11 in recombinant mammalian hosts, ayeast signal sequence is preferred for expression in yeast cells.Examples of suitable yeast signal sequences are those derived from yeastmating phermone α-factor (encoded by the MFαl gene), invertase (encodedby the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene). See,for example, Romanos et al., “Expression of Cloned Genes in Yeast,” inDNA Cloning 2: A Practical Approach, 2^(nd) Edition, Glover and Hames(eds.), pages 123-167 (Oxford University Press 1995).

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, Zkun11can be expressed as a fusion protein comprising a glutathioneS-transferase polypeptide. Glutathione S-transferease fusion proteinsare typically soluble, and easily purifiable from E. coli lysates onimmobilized glutathione columns. In similar approaches, a Zkun11 fusionprotein comprising a maltose binding protein polypeptide can be isolatedwith an amylose resin column, while a fusion protein comprising theC-terminal end of a truncated Protein A gene can be purified usingIgG-Sepharose. Established techniques for expressing a heterologouspolypeptide as a fusion protein in a bacterial cell are described, forexample, by Williams et al., “Expression of Foreign Proteins in E. coliUsing Plasmid Vectors and Purification of Specific PolyclonalAntibodies,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995). Inaddition, commercially available expression systems are available. Forexample, the PINPOINT Xa protein purification system (PromegaCorporation; Madison, Wis.) provides a method for isolating a fusionprotein comprising a polypeptide that becomes biotinylated duringexpression with a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolyHistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu—Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo).

Another form of fusion protein comprises a Zkun11 polypeptide and animmunoglobulin heavy chain constant region, typically an F_(c) fragment,which contains two constant region domains and a hinge region but lacksthe variable region. As an illustration, Chang et al., U.S. Pat. No.5,723,125, describe a fusion protein comprising a human interferon and ahuman immunoglobulin Fc fragment, in which the C-terminal of theinterferon is linked to the N-terminal of the Fc fragment by a peptidelinker moiety. An example of a peptide linker is a peptide comprisingprimarily a T cell inert sequence, which is immunologically inert. Anexemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGGS (SEQ ID NO:4). In such a fusion protein, an illustrative Fc moiety isa human γ4 chain, which is stable in solution and has little or nocomplement activating activity. Accordingly, the present inventioncontemplates a Zkun11 fusion protein that comprises a Zkun11 moiety anda human Fc fragment, wherein the C-terminus of the Zkun11 moiety isattached to the N-terminus of the Fc fragment via a peptide linker, suchas a peptide consisting of the amino acid sequence of SEQ ID NO:4. TheZkun11 moiety can be a Zkun11 molecule or a fragment thereof.

In another variation, a Zkun11 fusion protein comprises an IgG sequence,a Zkun11 moiety covalently joined to the aminoterminal end of the IgGsequence, and a signal peptide that is covalently joined to theaminoterminal of the Zkun11 moiety, wherein the IgG sequence consists ofthe following elements in the following order: a hinge region, a CH₂domain, and a CH₃ domain. Accordingly, the IgG sequence lacks a CH₁domain. The Zkun11 moiety displays a Zkun11 activity, as describedherein, such as the ability to bind with a Zkun11 antibody or theability to inhibit serine protease activity. This general approach toproducing fusion proteins that comprise both antibody and nonantibodyportions has been described by LaRochelle et al., EP 742830 (WO95/21258).

Fusion proteins comprising a Zkun11 moiety and an Fc moiety can be used,for example, as an in vitro assay tool. For example, the presence of aZkun11 target enzyme in a biological sample can be detected using aZkun11-antibody fusion protein, in which the Zkun11 moiety is used totarget the enzyme, and a macromolecule, such as Protein A or anti-Fcantibody, is used to detect the bound fusion protein-enzyme complex.Furthermore, such fusion proteins can be used to identify molecules thatinterfere with the binding of Zkun11 and a substrate for the cognateenzyme.

Moreover, using methods described in the art, hybrid Zkun11 proteins canbe constructed using regions or domains of the inventive Zkun11 incombination with those of other Kunitz inhibitor proteins, orheterologous proteins (see, for example, Picard, Cur. Opin. Biology5:511 (1994)). These methods allow the determination of the biologicalimportance of larger domains or regions in a polypeptide of interest.Such hybrids may alter reaction kinetics, binding, constrict or expandthe substrate specificity, or alter tissue and cellular localization ofa polypeptide, and can be applied to polypeptides of unknown structure.As an illustration, polypeptides can be devised that comprise a Zkun11polypeptide, or variant, thereof, and one or more Kunitz domainsseparated by amino acid linkers. Such linkers can comprise any aminoacids, so long as the higher order structure of the Kunitz domains isnot disrupted. For example, the linkers can comprise about 5, about 10,about 20, about 40, about 60, about 80, about 100, or more amino acidresidues.

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating the components. Alternatively, a polynucleotide encodingboth components of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. General methods for enzymatic and chemical cleavage of fusionproteins are described, for example, by Ausubel (1995) at pages 16-19 to16-25.

6. Zkun11 Analogs and Zkun11 Inhibitors

One general class of Zkun11 analogs are variants having an amino acidsequence that is a mutation of the amino acid sequence disclosed herein.Another general class of Zkun11 analogs is provided by anti-idiotypeantibodies, and fragments thereof, as described below. Moreover,recombinant antibodies comprising anti-idiotype variable domains can beused as analogs (see, for example, Monfardini et al., Proc. Assoc. Am.Physicians 108:420 (1996)). Since the variable domains of anti-idiotypeZkun11 antibodies mimic Zkun11, these domains can provide Zkun11activity. Methods of producing anti-idiotypic catalytic antibodies areknown to those of skill in the art (see, for example, Joron et al., Ann.N Y Acad. Sci. 672:216 (1992), Friboulet et al., Appl. Biochem.Biotechnol. 47:229 (1994), and Avalle et al., Ann. N Y Acad. Sci.864:118 (1998)).

Another approach to identifying Zkun11 analogs is provided by the use ofcombinatorial libraries. Methods for constructing and screening phagedisplay and other combinatorial libraries are provided, for example, byKay et al., Phage Display of Peptides and Proteins (Academic Press1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al., U.S. Pat. No.5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

Serine proteases can be used to produce labeled polypeptide fragmentsfrom a labeled protein substrate. Therefore, an illustrative in vitrouse of Zkun11 and its analogs is to control the generation of suchproteolysis cleavage products. Serine proteases are also used incleaning solutions, such as solutions to clean and to disinfect contactlenses (see, for example, Aaslyng et al., U.S. Pat. No. 5,985,629). Suchcleaning solutions also include protease inhibitors.

In addition, Zkun11 proteins may be used in the laboratory or commercialpreparation of proteins from cultured cells. The proteins can be usedalone to inhibit specific proteolysis or can be combined with otherproteinase inhibitors to provide a “cocktail” with a broad spectrum ofactivity. Of particular interest is the inhibition of cellularproteases, which can be release during cell lysis. Zkun11 proteins canalso be used in the laboratory as a tissue culture additive to preventcell detachment. Those of skill in the art can devise other uses formolecules having Zkun11 activity.

The activity of Zkun11 molecules of the present invention can bemeasured using a variety of assays that measure serine proteaseactivity. For example, Zkun11 activity can be assessed by measuringinhibition in a standard in vitro serine protease assay (see, forexample, Stief and Heimburger, U.S. Pat. No. 5,057,414 (1991)). Those ofskill in the art are aware of a variety of substrates suitable for invitro assays, such as Suc-Ala-Ala-Pro-Phe-pNA, fluoresceinmono-p-guanidinobenzoate hydrochloride,benzyloxycarbonyl-L-Arginyl-S-benzylester, Nalpha-Benzoyl-L-arginineethyl ester hydrochloride, and the like. In addition, protease assaykits available from commercial sources, such as Calbiochem® (San Diego,Calif.). For general references, see Barrett (Ed.), Methods inEnzymology, Proteolytic Enzymes: Serine and Cysteine Peptidases(Academic Press Inc. 1994), and Barrett et al., (Eds.), Handbook ofProteolytic Enzymes (Academic Press Inc. 1998).

Solution in vitro assays can be used to identify a Zkun11 target enzyme.Solid phase systems can also be used to identify a Zkun11 target enzyme.For example, a Zkun11 polypeptide or Zkun11 fusion protein can beimmobilized onto the surface of a receptor chip of a commerciallyavailable biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden).The use of this instrument is disclosed, for example, by Karlsson,Immunol. Methods 145:229 (1991), and Cunningham and Wells, J. Mol. Biol.234:554 (1993).

In brief, a Zkun11 polypeptide or fusion protein is covalently attached,using amine or sulfhydryl chemistry, to dextran fibers that are attachedto gold film within a flow cell. A test sample is then passed throughthe cell. If a Zkun11 target enzyme is present in the sample, it willbind to the immobilized polypeptide or fusion protein, causing a changein the refractive index of the medium, which is detected as a change insurface plasmon resonance of the gold film. This system allows thedetermination on- and off-rates, from which binding affinity can becalculated, and assessment of the stoichiometry of binding, as well asthe kinetic effects of Zkun11 mutation. This system can also be used toidentify other substrates for the Zkun11 target enzyme, and to examineantibody-antigen interactions, and the interactions of othercomplement/anti-complement pairs.

7. Production of Zkun11 Polypeptides in Cultured Cells

The polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion proteins, can be producedin recombinant host cells following conventional techniques. To expressa Zkun11 gene, a nucleic acid molecule encoding the polypeptide must beoperably linked to regulatory sequences that control transcriptionalexpression in an expression vector and then, introduced into a hostcell. In addition to transcriptional regulatory sequences, such aspromoters and enhancers, expression vectors can include translationalregulatory sequences and a marker gene, which is suitable for selectionof cells that carry the expression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, a Zkun11 expression vector maycomprise a Zkun11 gene and a secretory sequence derived from a Zkun11gene or another secreted gene.

Zkun11 proteins of the present invention may be expressed in mammaliancells. Examples of suitable mammalian host cells include African greenmonkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells(293 -HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570;ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin etal., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1;ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control Zkun11 gene expression inmammalian cells if the prokaryotic promoter is regulated by a eukaryoticpromoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman etal., Nucl. Acids Res. 19:4485 (1991)).

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. An exemplary amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins (e.g., CD4, CD8,Class I MHC, and placental alkaline phosphatase) may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

Zkun11 polypeptides can also be produced by cultured cells using a viraldelivery system. Exemplary viruses for this purpose include adenovirus,herpesvirus, vaccinia virus and adeno-associated virus (AAV).Adenovirus, a double-stranded DNA virus, is currently the best studiedgene transfer vector for delivery of heterologous nucleic acid (for areview, see Becker et al., Meth. Cell Biol. 43:161 (1994), and Douglasand Curiel, Science & Medicine 4:44 (1997)). Advantages of theadenovirus system include the accommodation of relatively large DNAinserts, the ability to grow to high-titer, the ability to infect abroad range of mammalian cell types, and flexibility that allows usewith a large number of available vectors containing different promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Forexample, adenovirus vector infected human 293 cells (ATCC Nos. CRL-1573,45504, 45505) can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

Zkun11 genes may also be expressed in other higher eukaryotic cells,such as avian, fungal, insect, yeast, or plant cells. The baculovirussystem provides an efficient means to introduce cloned Zkun11 genes intoinsect cells. Suitable expression vectors are based upon the Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV), and containwell-known promoters such as Drosophila heat shock protein (hsp) 70promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus p10 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the Zkun11 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, andRapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed Zkun11 polypeptide, for example, aGlu—Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952(1985)). Using a technique known in the art, a transfer vectorcontaining a Zkun11 gene is transformed into E. coli, and screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native Zkun11 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), orbaculovirus gp67 (PharMingen: San Diego, Calif.) can be used inconstructs to replace the native Zkun11 secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sj9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×1 cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). An illustrative vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which can be linearized prior to transformation. Forpolypeptide production in P. methanolica, the promoter and terminator inthe plasmid can be that of a P. methanolica gene, such as a P.methanolica alcohol utilization gene (AUG1 or AUG2). Other usefulpromoters include those of the dihydroxyacetone synthase (DHAS), formatedehydrogenase (FMD), and catalase (CAT) genes. To facilitate integrationof the DNA into the host chromosome, the entire expression segment ofthe plasmid can be flanked at both ends by host DNA sequences. Anillustrative selectable marker for use in Pichia methanolica is a P.methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazolecarboxylase (AIRC; EC 4.1.1.21), and which allows ade2 host cells togrow in the absence of adenine. For large-scale, industrial processeswhere it is desirable to minimize the use of methanol, host cells can beused in which both methanol utilization genes (AUG1 and AUG2) aredeleted. For production of secreted proteins, host cells can be usedthat are deficient in vacuolar protease genes (PEP4 and PRB1).Electroporation is used to facilitate the introduction of a plasmidcontaining DNA encoding a polypeptide of interest into P. methanolicacells. P. methanolica cells can be transformed by electroporation usingan exponentially decaying, pulsed electric field having a field strengthof from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a timeconstant (t) of from 1 to 40 milliseconds, most preferably about 20milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, Zkun11 genes can be expressed in prokaryotic host cells.Suitable promoters that can be used to express Zkun11 polypeptides in aprokaryotic host are well-known to those of skill in the art and includepromoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, theP_(R) and P_(L) promoters of bacteriophage lambda, the trp, recA, heatshock, lacUV5, tac, lpp-lacSpr, phoA, and lacZ promoters of E. coli,promoters of B. subtilis, the promoters of the bacteriophages ofBacillus, Streptomyces promoters, the int promoter of bacteriophagelambda, the bla promoter of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene. Prokaryotic promoters have beenreviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson et al.,Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and byAusubel et al. (1995).

Useful prokaryotic hosts include E. coli and Bacillus subtilus. Suitablestrains of E. coli include BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE,DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3, DH11S, C600,HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18,ER1451, and ER1647 (see, for example, Brown (ed.), Molecular BiologyLabfax (Academic Press 1991)). Suitable strains of Bacillus subtilusinclude BR151, YB886, MI119, MI120, and B170 (see, for example, Hardy,“Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach, Glover(ed.) (IRL Press 1985)).

When expressing a Zkun11 polypeptide in bacteria such as E. coli, thepolypeptide may be retained in the cytoplasm, typically as insolublegranules, or may be directed to the periplasmic space by a bacterialsecretion sequence. In the former case, the cells are lysed, and thegranules are recovered and denatured using, for example, guanidineisothiocyanate or urea. The denatured polypeptide can then be refoldedand dimerized by diluting the denaturant, such as by dialysis against asolution of urea and a combination of reduced and oxidized glutathione,followed by dialysis against a buffered saline solution. In the lattercase, the polypeptide can be recovered from the periplasmic space in asoluble and functional form by disrupting the cells (by, for example,sonication or osmotic shock) to release the contents of the periplasmicspace and recovering the protein, thereby obviating the need fordenaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), Severinov and Muir, J. Biol. Chem. 273:16205 (1998), andKochendoerfer and Kent, Curr. Opin. Chem. Biol. 3:665 (1999)).

8. Isolation of Zkun11 Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention may also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. Certain purified polypeptide preparationsare substantially free of other polypeptides, particularly otherpolypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of Zkun11 purified from natural sources, andrecombinant Zkun11 polypeptides and fusion Zkun11 polypeptides purifiedfrom recombinant host cells. In general, ammonium sulfate precipitationand acid or chaotrope extraction may be used for fractionation ofsamples. Exemplary purification steps may include hydroxyapatite, sizeexclusion, FPLC and reverse-phase high performance liquidchromatography. Suitable chromatographic media include derivatizeddextrans, agarose, cellulose, polyacrylamide, specialty silicas, and thelike. PEI, DEAE, QAE and Q derivatives are preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in Zkun11 isolation and purification can bedevised by those of skill in the art. For example, anti-Zkun11antibodies, obtained as described below, can be used to isolate largequantities of protein by immunoaffinity purification.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

Zkun11 polypeptides or fragments thereof may also be prepared throughchemical synthesis, as described above. Zkun11 polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; PEGylated ornon-PEGylated; and may or may not include an initial methionine aminoacid residue.

The present invention also contemplates chemically modified Zkun11compositions, in which a Zkun11 polypeptide is linked with a polymer.Typically, the polymer is water soluble so that the Zkun11 conjugatedoes not precipitate in an aqueous environment, such as a physiologicalenvironment. An example of a suitable polymer is one that has beenmodified to have a single reactive group, such as an active ester foracylation, or an aldehyde for alkylation, In this way, the degree ofpolymerization can be controlled. An example of a reactive aldehyde ispolyethylene glycol propionaldehyde, or mono-(C₁-C₁₀) alkoxy, or aryloxyderivatives thereof (see, for example, Harris, et al., U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. Moreover, amixture of polymers can be used to produce Zkun11 conjugates.

Zkun11 conjugates used for therapy can comprise pharmaceuticallyacceptable water-soluble polymer moieties. Suitable water-solublepolymers include polyethylene glycol (PEG), monomethoxy-PEG,mono-(C₁-C₁₀)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG,tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonatePEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, dextran, cellulose, or other carbohydrate-based polymers.Suitable PEG may have a molecular weight from about 600 to about 60,000,including, for example, 5,000, 12,000, 20,000 and 25,000. A Zkun11conjugate can also comprise a mixture of such water-soluble polymers.Anti-Zkun11 antibodies or anti-idiotype antibodies can also beconjugated with a water-soluble polymer.

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

Peptides and polypeptides of the present invention comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of SEQID NO:2. Within certain embodiments of the invention, the polypeptidescomprise 10, 20, 30, 40, or more contiguous residues of these amino acidsequences. Nucleic acid molecules encoding such peptides andpolypeptides are useful as polymerase chain reaction primers and probes.

In addition to the uses described above, polynucleotides andpolypeptides of the present invention are useful as educational tools inlaboratory practicum kits for courses related to genetics and molecularbiology, protein chemistry, and antibody production and analysis. Due toits unique polynucleotide and polypeptide sequences, molecules of Zkun11can be used as standards or as “unknowns” for testing purposes. Forexample, Zkun11 polynucleotides can be used as an aid, such as, forexample, to teach a student how to prepare expression constructs forbacterial, viral, or mammalian expression, including fusion constructs,wherein Zkun11 is the gene to be expressed; for determining therestriction endonuclease cleavage sites of the polynucleotides;determining mRNA and DNA localization of Zkun11 polynucleotides intissues (i.e., by northern and Southern blotting as well as polymerasechain reaction); and for identifying related polynucleotides andpolypeptides by nucleic acid hybridization. As an illustration, studentswill find that HinfI digestion of a nucleic acid molecule consisting ofthe nucleotide sequence of SEQ ID NO:1 provides fragments of about 48base pairs, and 105 base pairs.

Zkun11 polypeptides can be used as an aid to teach preparation ofantibodies; identifying proteins by western blotting; proteinpurification; determining the weight of expressed Zkun11 polypeptides asa ratio to total protein expressed; identifying peptide cleavage sites;coupling amino and carboxyl terminal tags; amino acid sequence analysis,as well as, but not limited to monitoring biological activities of boththe native and tagged protein (i.e., protease inhibition) in vitro andin vivo. For example, students will find that digestion ofunglycosylated Zkun11 with V8 in ammonium acetate yields fragmentshaving approximate molecular weights of 3256, 2442, and 380, whereasdigestion of unglycosylated Zkun11 with clostripain yields fragmentshaving approximate molecular weights of 1860, 2535, and 1682.

Zkun11 polypeptides can also be used to teach analytical skills such asmass spectrometry, circular dichroism, to determine conformation,especially of the four alpha helices, x-ray crystallography to determinethe three-dimensional structure in atomic detail, nuclear magneticresonance spectroscopy to reveal the structure of proteins in solution.For example, a kit containing the Zkun11 can be given to the student toanalyze. Since the amino acid sequence would be known by the instructor,the protein can be given to the student as a test to determine theskills or develop the skills of the student, the instructor would thenknow whether or not the student has correctly analyzed the polypeptide.Since every polypeptide is unique, the educational utility of Zkun11would be unique unto itself.

The antibodies which bind specifically to Zkun11 can be used as ateaching aid to instruct students how to prepare affinity chromatographycolumns to purify Zkun11, cloning and sequencing the polynucleotide thatencodes an antibody and thus as a practicum for teaching a student howto design humanized antibodies. The Zkun11 gene, polypeptide, orantibody would then be packaged by reagent companies and sold toeducational institutions so that the students gain skill in art ofmolecular biology. Because each gene and protein is unique, each geneand protein creates unique challenges and learning experiences forstudents in a lab practicum. Such educational kits containing the Zkun11gene, polypeptide, or antibody are considered within the scope of thepresent invention.

9. Production of Antibodies to Zkun11 Proteins

Antibodies to Zkun11 can be obtained, for example, using as an antigenthe product of a Zkun11 expression vector or Zkun11 isolated from anatural source. Particularly useful anti-Zkun11 antibodies “bindspecifically” with Zkun11. Antibodies are considered to be specificallybinding if the antibodies exhibit at least one of the following twoproperties: (1) antibodies bind to Zkun11 with a threshold level ofbinding activity, and (2) antibodies do not significantly cross-reactwith polypeptides related to Zkun11.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a Zkun11 polypeptide, peptide or epitope with a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). With regard to the secondcharacteristic, antibodies do not significantly cross-react with relatedpolypeptide molecules, for example, if they detect Zkun11, but not knownrelated polypeptides using a standard Western blot analysis. Examples ofknown related polypeptides are orthologs and proteins from the samespecies that are members of a protein family. For example,specifically-binding anti-Zkun11 antibodies bind with Zkun11, but notwith known Kunitz inhibitor proteins.

Anti-Zkun11 antibodies can be produced using antigenic Zkun11epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, or between 15 to about 30 amino acids contained withinSEQ ID NO:2. However, peptides or polypeptides comprising a largerportion of an amino acid sequence of the invention, containing from 30to 40 amino acids, or any length up to and including the entire aminoacid sequence of a polypeptide of the invention, also are useful forinducing antibodies that bind with Zkun11. It is desirable that theamino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues, while hydrophobic residues arepreferably avoided). Moreover, amino acid sequences containing prolineresidues may be also be desirable for antibody production.

As an illustration, potential antigenic sites in Zkun11 were identifiedusing the Jameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988),as implemented by the PROTEAN program (version 3.14) of LASERGENE(DNASTAR; Madison, Wis.). Default parameters were used in this analysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Gamier-Robson, Gamier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Gamier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors were combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function was applied to the antigenic index,which broadens major surface peaks by adding 20, 40, 60, or 80% of therespective peak value to account for additional free energy derived fromthe mobility of surface regions relative to interior regions. Thiscalculation was not applied, however, to any major peak that resides ina helical region, since helical regions tend to be less flexible.

The results of this analysis indicated that amino acid residues 4 to 10(“antigenic peptide 1”), and amino acid residues 35 to 43 (“antigenicpeptide 2”), of SEQ ID NO:2 would provide suitable antigenic peptides.The present invention contemplates the use of any one of antigenicpeptides 1 and 2 to generate antibodies to Zkun11. The present inventionalso contemplates polypeptides comprising at least one of theseantigenic peptides.

Polyclonal antibodies to recombinant Zkun11 protein or to Zkun11isolated from natural sources can be prepared using methods well-knownto those of skill in the art. Antibodies can also be generated using aZkun11-glutathione transferase fusion protein, which is similar to amethod described by Burrus and McMahon, Exp. Cell. Res. 220:363 (1995).General methods for producing polyclonal antibodies are described, forexample, by Green et al., “Production of Polyclonal Antisera,” inImmunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992),and Williams et al., “Expression of foreign proteins in E. coli usingplasmid vectors and purification of specific polyclonal antibodies,” inDNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),page 15 (Oxford University Press 1995).

The immunogenicity of a Zkun11 polypeptide can be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of Zkun11 or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like,” suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLM), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorse, cow, dog, chicken, rat, mouse, rabbit, goat, guinea pig, orsheep, an anti-Zkun11 antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310(1990).

Alternatively, monoclonal anti-Zkun11 antibodies can be generated.Rodent monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art (see, for example, Kohler etal., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a Zkun11 gene product, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-Zkun11 antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal, Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-Zkun11 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem.J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains which are connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97 (1991) (also see, Bird et al.,Science 242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271 (1993), and Sandhu, supra).

As an illustration, a scFV can be obtained by exposing lymphocytes toZkun11 polypeptide in vitro, and selecting antibody display libraries inphage or similar vectors (for instance, through use of immobilized orlabeled Zkun11 protein or peptide). Genes encoding polypeptides havingpotential Zkun11 polypeptide binding domains can be obtained byscreening random peptide libraries displayed on phage (phage display) oron bacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al., U.S. Pat.No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kayet al., Phage Display of Peptides and Proteins (Academic Press, Inc.1996)) and random peptide display libraries and kits for screening suchlibraries are available commercially, for instance from CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the Zkun11 sequences disclosed herein to identifyproteins which bind to Zkun11.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-Zkun11 antibody may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies are produced bytransferring mouse complementary determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain. Typical residues of human antibodies are then substituted in theframework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are described, for example, by Orlandi et al., Proc. Nat'l Acad.Sci. USA 86:3833 (1989). Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321:522(1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992),Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J. Immun.150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols (HumanaPress, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-Zkun11 antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypeantibodies can be prepared using anti-Zkun11 antibodies or antibodyfragments as immunogens with the techniques, described above. As anotheralternative, humanized anti-idiotype antibodies or subhuman primateanti-idiotype antibodies can be prepared using the above-describedtechniques. Methods for producing anti-idiotype antibodies aredescribed, for example, by Irie, U.S. Pat. No. 5,208,146, Greene, et.al., U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.77:1875 (1996).

Anti-idiotype Zkun11 antibodies, as well as Zkun11 polypeptides. can beused to identify and to isolate Zkun11 substrates and inhibitors. Forexample, proteins and peptides of the present invention can beimmobilized on a column and used to bind substrate and inhibitorproteins from biological samples that are run over the column (Hermansonet al. (eds.), Immobilized Affinity Ligand Techniques, pages 195-202(Academic Press 1992)). Radiolabeled or affinity labeled Zkun11polypeptides can also be used to identify or to localize Zkun11substrates and inhibitors in a biological sample (see, for example,Deutscher (ed.), Methods in Enzymol., vol. 182, pages 721-37 (AcademicPress 1990); Brunner et al., Ann. Rev. Biochem. 62:483 (1993); Fedan etal., Biochem. Pharmacol. 33:1167 (1984)).

10. Use of Zkun11 Nucleotide Sequences to Detect Zkun11 Gene Expressionand to Examine Zkun11 Gene Structure

Nucleic acid molecules can be used to detect the expression of a Zkun11gene in a biological sample. Such probe molecules includedouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NO:1, or a fragment thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:1, or a portion thereof. As usedherein, the term “portion” refers to at least eight nucleotides to atleast 20 or more nucleotides. Probe molecules may be DNA, RNA,oligonucleotides, and the like. Certain probes bind with regions of aZkun11 gene that have a low sequence similarity to comparable regions inother serine protease inhibitors.

In a basic assay, a single-stranded probe molecule is incubated withRNA, isolated from a biological sample, under conditions of temperatureand ionic strength that promote base pairing between the probe andtarget Zkun11 RNA species. After separating unbound probe fromhybridized molecules, the amount of hybrids is detected.

Well-established hybridization methods of RNA detection include northernanalysis and dot/slot blot hybridization (see, for example, Ausubel(1995) at pages 4-1 to 4-27, and Wu et al. (eds.), “Analysis of GeneExpression at the RNA Level,” in Methods in Gene Biotechnology, pages225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectablylabeled with radioisotopes such as ³²P or ³⁵S. Alternatively, Zkun11 RNAcan be detected with a nonradioactive hybridization method (see, forexample, Isaac (ed.), Protocols for Nucleic Acid Analysis byNonradioactive Probes (Humana Press, Inc. 1993)). Typically,nonradioactive detection is achieved by enzymatic conversion ofchromogenic or chemiluminescent substrates. Illustrative nonradioactivemoieties include biotin, fluorescein, and digoxigenin.

Zkun11 oligonucleotide probes are also useful for in vivo diagnosis. Asan illustration, ¹⁸F-labeled oligonucleotides can be administered to asubject and visualized by positron emission tomography (Tavitian et al.,Nature Medicine 4:467 (1998)).

Numerous diagnostic procedures take advantage of the polymerase chainreaction (PCR) to increase sensitivity of detection methods. Standardtechniques for performing PCR are well-known (see, generally, Mathew(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),White (ed.), PCR Protocols: Current Methods and Applications (HumanaPress, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (HumanaPress, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (HumanaPress, Inc. 1998)).

One variation of PCR for diagnostic assays is reverse transcriptase-PCR(RT-PCR). In the RT-PCR technique, RNA is isolated from a biologicalsample, reverse transcribed to cDNA, and the cDNA is incubated withZkun11 primers (see, for example, Wu et al. (eds.), “Rapid Isolation ofSpecific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages15-28 (CRC Press, Inc. 1997)). PCR is then performed and the productsare analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, forexample, the guanidinium-thiocyanate cell lysis procedure describedabove. Alternatively, a solid-phase technique can be used to isolatemRNA from a cell lysate. A reverse transcription reaction can be primedwith the isolated RNA using random oligonucleotides, short homopolymersof dT, or Zkun11 anti-sense oligomers. Oligo-dT primers offer theadvantage that various mRNA nucleotide sequences are amplified that canprovide control target sequences. Zkun11 sequences are amplified by thepolymerase chain reaction using two flanking oligonucleotide primersthat are typically 20 bases in length.

PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled Zkun11 probe, andexamined by autoradiography. Additional alternative approaches includethe use of digoxigenin-labeled deoxyribonucleic acid triphosphates toprovide chemiluminescence detection, and the C-TRAK calorimetric assay.

Another approach for detection of Zkun11 expression is cycling probetechnology, in which a single-stranded DNA target binds with an excessof DNA-RNA-DNA chimeric probe to form a complex, the RNA portion iscleaved with RNAase H, and the presence of cleaved chimeric probe isdetected (see, for example, Beggs et al., J. Clin. Microbiol. 34:2985(1996), Bekkaoui et al., Biotechniques 20:240 (1996)). Alternativemethods for detection of Zkun11 sequences can utilize approaches such asnucleic acid sequence-based amplification, cooperative amplification oftemplates by cross-hybridization, and the ligase chain reaction (see,for example, Marshall et al., U.S. Pat. No. 5,686,272 (1997), Dyer etal., J. Virol. Methods 60:161 (1996), Ehricht et al., Eur. J. Biochem.243:358 (1997), and Chadwick et al., J. Virol. Methods 70:59 (1998)).Other standard methods are known to those of skill in the art.

Zkun11 probes and primers can also be used to detect and to localizeZkun11 gene expression in tissue samples. Methods for such in situhybridization are well-known to those of skill in the art (see, forexample, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc.1994), Wu et al. (eds.), “Analysis of Cellular DNA or Abundance of mRNAby Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, pages279-289 (CRC Press, Inc. 1997)). Various additional diagnosticapproaches are well-known to those of skill in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (HumanaPress, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases(Humana Press, Inc., 1996)).

Zkun11 nucleotide sequences can also be used in linkage-based testingfor various diseases, and to determine whether a subject's chromosomescontain a mutation in the Zkun11 gene. Detectable chromosomalaberrations at the Zkun11 gene locus include, but are not limited to,aneuploidy, gene copy number changes, insertions, deletions, restrictionsite changes and rearrangements. Of particular interest are geneticalterations that inactivate a Zkun11 gene.

Aberrations associated with a Zkun11 locus can be detected using nucleicacid molecules of the present invention by employing molecular genetictechniques, such as restriction fragment length polymorphism analysis,short tandem repeat analysis employing PCR techniques,amplification-refractory mutation system analysis, single-strandconformation polymorphism detection, RNase cleavage methods, denaturinggradient gel electrophoresis, fluorescence-assisted mismatch analysis,and other genetic analysis techniques known in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis,Molecular Diagnostics (Human Press, Inc. 1996), Elles (ed.) MolecularDiagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren(ed.), Laboratory Protocols for Mutation Detection (Oxford UniversityPress 1996), Birren et al. (eds.), Genome Analysis, Vol. 2: DetectingGenes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al.(eds.), Current Protocols in Human Genetics (John Wiley & Sons 1998),and Richards and Ward, “Molecular Diagnostic Testing,” in Principles ofMolecular Medicine, pages 83-88 (Humana Press, Inc. 1998)).

The protein truncation test is also useful for detecting theinactivation of a gene in which translation-terminating mutationsproduce only portions of the encoded protein (see, for example,Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to thisapproach, RNA is isolated from a biological sample, and used tosynthesize cDNA. PCR is then used to amplify the Zkun11 target sequenceand to introduce an RNA polymerase promoter, a translation initiationsequence, and an in-frame ATG triplet. PCR products are transcribedusing an RNA polymerase, and the transcripts are translated in vitrowith a T7-coupled reticulocyte lysate system. The translation productsare then fractionated by SDS-PAGE to determine the lengths of thetranslation products. The protein truncation test is described, forexample, by Dracopoli et al. (eds.), Current Protocols in HumanGenetics, pages 9.11.1-9.11.18 (John Wiley & Sons 1998).

The chromosomal location of the Zkun11 gene can be determined usingradiation hybrid mapping, which is a somatic cell genetic techniquedeveloped for constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245 (1990)). Partial or fullknowledge of a gene's sequence allows one to design PCR primers suitablefor use with chromosomal radiation hybrid mapping panels. Radiationhybrid mapping panels are commercially available which cover the entirehuman genome, such as the Stanford G3 RH Panel and the Gene Bridge 4 RHPanel (Research Genetics, Inc., Huntsville, Ala.). These panels enablerapid, PCR-based chromosomal localizations and ordering of genes,sequence-tagged sites (STSs), and other nonpolymorphic and polymorphicmarkers within a region of interest. This includes establishing directlyproportional physical distances between newly discovered genes ofinterest and previously mapped markers.

The present invention also contemplates kits for performing a diagnosticassay for Zkun11 gene expression or to analyze the Zkun11 locus of asubject. Such kits comprise nucleic acid probes, such as double-strandednucleic acid molecules comprising the nucleotide sequence of SEQ IDNO:1, or a fragment thereof, as well as single-stranded nucleic acidmolecules having the complement of the nucleotide sequence of SEQ IDNO:1, or a fragment thereof. Probe molecules may be DNA, RNA,oligonucleotides, and the like. Kits may comprise nucleic acid primersfor performing PCR.

Such a kit can contain all the necessary elements to perform a nucleicacid diagnostic assay described above. A kit will comprise at least onecontainer comprising a Zkun11 probe or primer. The kit may also comprisea second container comprising one or more reagents capable of indicatingthe presence of Zkun11 sequences. Examples of such indicator reagentsinclude detectable labels such as radioactive labels, fluorochromes,chemiluminescent agents, and the like. A kit may also comprise a meansfor conveying to the user that the Zkun11 probes and primers are used todetect Zkun11 gene expression. For example, written instructions maystate that the enclosed nucleic acid molecules can be used to detecteither a nucleic acid molecule that encodes Zkun11, or a nucleic acidmolecule having a nucleotide sequence that is complementary to aZkun11-encoding nucleotide sequence, or to analyze chromosomal sequencesassociated with the Zkun11 locus. The written material can be applieddirectly to a container, or the written material can be provided in theform of a packaging insert.

11. Use of Anti-Zkun11 Antibodies to Detect Zkun11 Protein

The present invention contemplates the use of anti-Zkun11 antibodies toscreen biological samples in vitro for the presence of Zkun11. In onetype of in vitro assay, anti-Zkun11 antibodies are used in liquid phase.For example, the presence of Zkun11 in a biological sample can be testedby mixing the biological sample with a trace amount of labeled Zkun11and an anti-Zkun11 antibody under conditions that promote bindingbetween Zkun11 and its antibody. Complexes of Zkun11 and anti-Zkun11 inthe sample can be separated from the reaction mixture by contacting thecomplex with an immobilized protein which binds with the antibody, suchas an Fc antibody or Staphylococcus protein A. The concentration ofZkun11 in the biological sample will be inversely proportional to theamount of labeled Zkun11 bound to the antibody and directly related tothe amount of free labeled Zkun11. Illustrative biological samplesinclude blood, urine, saliva, tissue biopsy, and autopsy material.

Alternatively, in vitro assays can be performed in which anti-Zkun11antibody is bound to a solid-phase carrier. For example, antibody can beattached to a polymer, such as aminodextran, in order to link theantibody to an insoluble support such as a polymer-coated bead, a plateor a tube. Other suitable in vitro assays will be readily apparent tothose of skill in the art.

In another approach, anti-Zkun11 antibodies can be used to detect Zkun11in tissue sections prepared from a biopsy specimen. Such immunochemicaldetection can be used to determine the relative abundance of Zkun11 andto determine the distribution of Zkun11 in the examined tissue. Generalimmunochemistry techniques are well established (see, for example,Ponder, “Cell Marking Techniques and Their Application,” in MammalianDevelopment: A Practical Approach, Monk (ed.), pages 115-38 (IRL Press1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods InMolecular Biology, Vol.10: Immunochemical Protocols (The Humana Press,Inc. 1992)).

Immunochemical detection can be performed by contacting a biologicalsample with an anti-Zkun11 antibody, and then contacting the biologicalsample with a detectably labeled molecule which binds to the antibody.For example, the detectably labeled molecule can comprise an antibodymoiety that binds to anti-Zkun11 antibody.

Alternatively, the anti-Zkun11 antibody can be conjugated withavidin/streptavidin (or biotin) and the detectably labeled molecule cancomprise biotin (or avidin/streptavidin). Numerous variations of thisbasic technique are well-known to those of skill in the art.

Alternatively, an anti-Zkun11 antibody can be conjugated with adetectable label to form an anti-Zkun11 immunoconjugate. Suitabledetectable labels include, for example, a radioisotope, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescent labelor colloidal gold. Methods of making and detecting suchdetectably-labeled immunoconjugates are well-known to those of ordinaryskill in the art, and are described in more detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-Zkun11 immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, anti-Zkun11 immunoconjugates can be detectably labeled bycoupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged immunoconjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-Zkun11immunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, anti-Zkun11 immunoconjugates can be detectably labeled bylinking an anti-Zkun11 antibody component to an enzyme. When theanti-Zkun11 -enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety, which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels, which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-Zkun11 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al., Clin. Chim. Acta 70:1 (1976), Schurs etal., Clin. Chim. Acta 81:1 (1977), Shih et al., Int'l J. Cancer 46:1101(1990), Stein et al., Cancer Res. 50:1330 (1990), and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-Zkun11 antibodies that have beenconjugated with avidin, streptavidin, and biotin (see, for example,Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods InEnzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,“Immunochemical Applications of Avidin-Biotin Technology,” in Methods InMolecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The HumanaPress, Inc. 1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

In a related approach, biotin- or FITC-labeled Zkun11 can be used toidentify cells that bind Zkun11. Such can binding can be detected, forexample, using flow cytometry.

The present invention also contemplates kits for performing animmunological diagnostic assay for Zkun11 gene expression. Such kitscomprise at least one container comprising an anti-Zkun11 antibody, orantibody fragment. A kit may also comprise a second container comprisingone or more reagents capable of indicating the presence of Zkun11antibody or antibody fragments. Examples of such indicator reagentsinclude detectable labels such as a radioactive label, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescentlabel, colloidal gold, and the like. A kit may also comprise a means forconveying to the user that Zkun11 antibodies or antibody fragments areused to detect Zkun11 protein. For example, written instructions maystate that the enclosed antibody or antibody fragment can be used todetect Zkun11. The written material can be applied directly to acontainer, or the written material can be provided in the form of apackaging insert.

12. Therapeutic Uses of Polypeptides Having Zkun11 Activity

The present invention includes the use of proteins, polypeptides, andpeptides having Zkun11 activity (such as Zkun11 polypeptides,anti-idiotype anti-Zkun11 antibodies, and Zkun11 fusion proteins) to asubject who lacks an adequate amount of this serine protease inhibitor.These molecules can be administered to any subject in need of treatment,and the present invention contemplates both veterinary and humantherapeutic uses. Illustrative subjects include mammalian subjects, suchas farm animals, domestic animals, and human patients.

For example, the Zkun11 proteins are contemplated for use in thetreatment or prevention of conditions associated with excessiveproteinase activity, such as an excess of trypsin, chymotrypsins A andB, plasmin, subtilisin, enterokinase, kallikrein, elastase, cathepsin G,acrosin, proteinase-3, thrombin, factor VIIa, factor IXa, factor Xa,factor XIa, factor XIIa, or matrix metalloproteinases. Such conditionsinclude, but are not limited to, acute pancreatitis, cardiopulmonarybypass-induced pulmonary injury, allergy-induced protease release, deepvein thrombosis, myocardial infarction, shock (including septic shock),hyperfibrinolytic hemorrhage, emphysema, rheumatoid arthritis, adultrespiratory distress syndrome, chronic inflammatory bowel disease,psoriasis, and other inflammatory conditions. Zkun11 proteins are alsocontemplated for use in preservation of platelet function, organpreservation, and wound healing.

Zkun11 proteins may be useful in the treatment of conditions arisingfrom an imbalance in hemostasis, including acquired coagulopathies,primary fibrinolysis and fibrinolysis due to cirrhosis, andcomplications from high-dose thrombolytic therapy. Acquiredcoagulopathies can result from liver disease, uremia, acute disseminatedintravascular coagulation, post-cardiopulmonary bypass, massivetransfusion, or Warfarin overdose (Humphries, Transfusion Medicine1:1181 (1994)). A deficiency or dysfunction in any of the procoagulantmechanisms predisposes a subject to either spontaneous hemorrhage orexcess blood loss associated with trauma or surgery. Acquiredcoagulopathies usually involve a combination of deficiencies, such asdeficiencies of a plurality of coagulation factors, or plateletdysfunction. In addition, subjects with liver disease commonlyexperience increased fibrinolysis due to an inability to maintain normallevels of α₂-antiplasmin or decreased hepatic clearance of plasminogenactivators (Shuman, “Hemorrhagic Disorders,” in Bennet and Plum (Eds.),Cecil Textbook of Medicine, 20th Ed. (W. B. Saunders Co. 1996)).

Primary fibrinolysis results from a massive release of plasminogenactivator. Conditions associated with primary fibrinolysis includecarcinoma of the prostate, acute promyelocytic leukemia, hemangiomas,and sustained release of plasminogen activator by endothelial cells dueto injection of venoms. The condition becomes critical when enoughplasmin is activated to deplete the circulating level of α₂-antiplasmin(Shuman, “Hemorrhagic Disorders,” in Bennet and Plum (Eds.), CecilTextbook of Medicine, 20th Ed. (W. B. Saunders Co. 1996)). Data suggestthat plasmin on endothelial cells may be related to the pathophysiologyof bleeding or rethrombosis observed in patients undergoing high-dosethrombolytic therapy for thrombosis. Plasmin may cause further damage tothe thrombogenic surface of blood vessels after thrombolysis, which mayresult in rethrombosis (Okajima, J. Lab. Clin. Med. 126:1377 (1995)).

Additional antithrombotic uses of Zkun11 proteins include treatment orprevention of deep vein thrombosis, pulmonary embolism, andpost-surgical thrombosis.

Zkun11 proteins may also be used within methods for inhibiting bloodcoagulation in mammals, such as in the treatment of disseminatedintravascular coagulation. In this way, Zkun11 proteins can substitutefor anticoagulants such as heparin, coumarin, and anti-thrombin III.Such methods will generally include administration of the protein in anamount sufficient to produce a clinically significant inhibition ofblood coagulation. Such amounts will vary with the nature of thecondition to be treated, but can be predicted on the basis of knownassays and experimental animal models.

Zkun11 proteins may also find therapeutic use in the blockage ofproteolytic tissue degradation. Proteolysis of extracellular matrix,connective tissue, and other tissues and organs is an element of manydiseases. This tissue destruction is believed to be initiated whenplasmin activates one or more matrix metalloproteinases (e.g.,collagenase and metallo-elastases). Inhibition of plasmin by Zkun11proteins may thus be beneficial in the treatment of these conditions.

Matrix metalloproteinases (MMPs) are believed to play a role inmetastases of cancers, abdominal aortic aneurysm, multiple sclerosis,rheumatoid arthritis, osteoarthritis, trauma and hemorrhagic shock, andcornial ulcers. MMPs produced by tumor cells break down and remodeltissue matrices during the process of metastatic spread. There isevidence to suggest that MMP inhibitors block this activity (Brown,Advan. Enzyme Regul. 35:293 (1995)). Abdominal aortic aneurysm ischaracterized by the degradation of extracellular matrix and loss ofstructural integrity of the aortic wall. Data suggest that plasmin maybe important in the sequence of events leading to this destruction ofaortic matrix (Jean-Claude et al., Surgery 116:472 (1994)). Proteolyticenzymes are also believed to contribute to the inflammatory tissuedamage of multiple sclerosis (Gijbels, J. Clin. Invest. 94:2177 (1994)).Rheumatoid arthritis is a chronic, systemic inflammatory diseasepredominantly affecting joints and other connective tissues, whereinproliferating inflammatory tissue (panus) may cause joint deformitiesand dysfunction (see, Arnett, in Cecil Textbook of Medicine, ibid.).Osteoarthritis is a chronic disease causing deterioration of the jointcartilage and other joint tissues and the formation of new bone (bonespurs) at the margins of the joints. There is evidence that MMPsparticipate in the degradation of collagen in the matrix ofosteoarthritic articular cartilage. Inhition of MMPs results in theinhibition of the removal of collagen from cartilage matrix (Spirito,Inflam. Res. 44 (supp. 2):S131 (1995); O'Byrne, Inflam. Res. 44 (supp.2):S117 (1995); Karran, Ann. Rheumatic Disease 54:662 (1995)).

Zkun11 proteins may also be useful in the treatment of trauma andhemorrhagic shock. Data suggest that administration of an MMP inhibitorafter hemorrhage improves cardiovascular response, hepatocellularfunction, and microvascular blood flow in various organs (Wang, Shock6:377 (1996)). Corneal ulcers, which can result in blindness, manifestas a breakdown of the collagenous stromal tissue. Damage due to thermalor chemical injury to corneal surfaces often results in a chronicwound-healing situation. There is direct evidence for the role of MMPsin basement membrane defects associated with failure to re-epithelializein cornea or skin (Fini, Am. J. Pathol. 149:1287 (1996)).

The Zkun11 proteins of the present invention may be combined with othertherapeutic agents to augment the activity (e.g., antithrombotic oranticoagulant activity) of such agents. For example, a Zkun11 proteinmay be used in combination with tissue plasminogen activator inthrombolytic therapy.

Generally, the dosage of administered polypeptide, protein or peptidewill vary depending upon such factors as the subject's age, weight,height, sex, general medical condition and previous medical history.Typically, it is desirable to provide the recipient with a dosage of amolecule having Zkun11 activity, which is in the range of from about 10μg/kg to 10 mg/kg body weight, 100 μg/kg to 5 mg/kg body weight, or 100μg/kg to 1 mg/kg body weight, although a lower or higher dosage also maybe administered as circumstances dictate.

Administration of a molecule having Zkun11 activity to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having Zkun11 activity can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thetherapeutic proteins are combined in a mixture with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient patient. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers arewell-known to those in the art. See, for example, Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995).

For purposes of therapy, molecules having Zkun11 activity and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having Zkun11 activity and a pharmaceuticallyacceptable carrier is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient patient.

A pharmaceutical composition comprising molecules having Zkun11 activitycan be furnished in liquid form, or in solid form. Liquid forms,including liposome-encapsulated formulations, are illustrated byinjectable solutions and oral suspensions. Exemplary solid forms includecapsules, tablets, and controlled-release forms, such as a miniosmoticpump or an implant. Other dosage forms can be devised by those skilledin the art, as shown, for example, by Ansel and Popovich, PharmaceuticalDosage Forms and Drug Delivery Systems, 5^(th) Edition (Lea & Febiger1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19^(th)Edition (Mack Publishing Company 1995), and by Ranade and Hollinger,Drug Delivery Systems (CRC Press 1996).

As an illustration, Zkun11 pharmaceutical compositions may be suppliedas a kit comprising a container that comprises Zkun11. Zkun11 can beprovided in the form of an injectable solution for single or multipledoses, or as a sterile powder that will be reconstituted beforeinjection. Such a kit may further comprise written information onindications and usage of the pharmaceutical composition. Moreover, suchinformation may include a statement that the Zkun11 composition iscontraindicated in patients with known hypersensitivity to Zkun11.

13. Therapeutic Uses of Zkun11 Nucleotide Sequences

The present invention includes the use of Zkun11 nucleotide sequences toprovide Zkun11 to a subject in need of such treatment, as discussedabove. In addition, a therapeutic expression vector can be provided thatinhibits Zkun11 gene expression, such as an anti-sense molecule, aribozyme, or an external guide sequence molecule.

There are numerous approaches to introduce a Zkun11 gene to a subject,including the use of recombinant host cells that express Zkun11,delivery of naked nucleic acid encoding Zkun11, use of a cationic lipidcarrier with a nucleic acid molecule that encodes Zkun11, and the use ofviruses that express Zkun1, such as recombinant retroviruses,recombinant adeno-associated viruses, recombinant adenoviruses, andrecombinant Herpes simplex viruses (see, for example, Mulligan, Science260:926 (1993), Rosenberg et al., Science 242:1575 (1988), LaSalle etal., Science 259:988 (1993), Wolff et al., Science 247:1465 (1990),Breakfield and Deluca, The New Biologist 3:203 (1991)). In an ex vivoapproach, for example, cells are isolated from a subject, transfectedwith a vector that expresses a Zkun11 gene, and then transplanted intothe subject.

In order to effect expression of a Zkun11 gene, an expression vector isconstructed in which a nucleotide sequence encoding a Zkun11 gene isoperably linked to a core promoter, and optionally a regulatory element,to control gene transcription. The general requirements of an expressionvector are described above.

Alternatively, a Zkun11 gene can be delivered using recombinant viralvectors, including for example, adenoviral vectors (e.g., Kass-Eisler etal., Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al., Proc.Nat'l Acad. Sci. USA 91:215 (1994), Li et al., Hum. Gene Ther. 4:403(1993), Vincent et al., Nat. Genet. 5:130 (1993), and Zabner et al.,Cell 75:207 (1993)), adenovirus-associated viral vectors (Flotte et al.,Proc. Nat'l Acad. Sci. USA 90:10613 (1993)), alphaviruses such asSemliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat. Nos.4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors(Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus vectors(Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali andPaoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)), pox viruses, suchas canary pox virus or vaccinia virus (Fisher-Hoch et al., Proc. Nat'lAcad. Sci. USA 86:317 (1989), and Flexner et al., Ann. N. Y. Acad. Sci.569:86 (1989)), and retroviruses (e.g., Baba et al., J. Neurosurg 79:729(1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J.Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993),Vile and Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S.Pat. No. 5,399,346). Within various embodiments, either the viral vectoritself, or a viral particle which contains the viral vector may beutilized in the methods and compositions described below.

As an illustration of one system, adenovirus, a double-stranded DNAvirus, is a well-characterized gene transfer vector for delivery of aheterologous nucleic acid molecule (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genescan be used to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1-deleted, and in addition, contain deletions of E2Aor E4 (Lusky et al., J. Virol. 72:2022 (1998); Raper et al., Human GeneTherapy 9:671 (1998)). The deletion of E2b has also been reported toreduce immune responses (Amalfitano et al., J. Virol. 72:926 (1998)). Bydeleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses, where all viral genes are deleted, are particularlyadvantageous for insertion of large inserts of heterologous DNA (for areview, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant HSV can be prepared in Verocells, as described by Brandt et al., J. Gen. Virol. 72:2043 (1991),Herold et al., J. Gen. Virol. 75:1211 (1994), Visalli and Brandt,Virology 185:419 (1991), Grau et al., Invest. Ophthalmol. Vis. Sci.30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992), and byBrown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).

Alternatively, an expression vector comprising a Zkun11 gene can beintroduced into a subject's cells by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al., Proc. Nat'lAcad. Sci. USA 85:8027 (1988)). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Liposomes can be used to direct transfection to particularcell types, which is particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides (e.g., hormones or neurotransmitters),proteins such as antibodies, or non-peptide molecules can be coupled toliposomes chemically.

Electroporation is another alternative mode of administration of aZkun11 nucleic acid molecules. For example, Aihara and Miyazaki, NatureBiotechnology 16:867 (1998), have demonstrated the use of in vivoelectroporation for gene transfer into muscle.

In an alternative approach to gene therapy, a therapeutic gene mayencode a Zkun11 anti-sense RNA that inhibits the expression of Zkun11.Methods of preparing anti-sense constructs are known to those in theart. See, for example, Erickson et al., Dev. Genet. 14:274 (1993)[transgenic mice], Augustine et al., Dev. Genet. 14:500 (1993) [murinewhole embryo culture], and Olson and Gibo, Exp. Cell Res. 241:134 (1998)[cultured cells]. Suitable sequences for Zkun11 anti-sense molecules canbe derived from the nucleotide sequences of Zkun11 disclosed herein.

Alternatively, an expression vector can be constructed in which aregulatory element is operably linked to a nucleotide sequence thatencodes a ribozyme. Ribozymes can be designed to express endonucleaseactivity that is directed to a certain target sequence in a mRNAmolecule (see, for example, Draper and Macejak, U.S. Pat. No. 5,496,698,McSwiggen, U.S. Pat. No. 5,525,468, Chowrira and McSwiggen, U.S. Pat.No. 5,631,359, and Robertson and Goldberg, U.S. Pat. No. 5,225,337). Inthe context of the present invention, ribozymes include nucleotidesequences that bind with Zkun11 mRNA.

In another approach, expression vectors can be constructed in which aregulatory element directs the production of RNA transcripts capable ofpromoting RNase P-mediated cleavage of mRNA molecules that encode aZkun11 gene. According to this approach, an external guide sequence canbe constructed for directing the endogenous ribozyme, RNase P, to aparticular species of intracellular mRNA, which is subsequently cleavedby the cellular ribozyme (see, for example, Altman et al., U.S. Pat. No.5,168,053, Yuan et al., Science 263:1269 (1994), Pace et al.,international publication No. WO 96/18733, George et al., internationalpublication No. WO 96/21731, and Werner et al., internationalpublication No. WO 97/33991). Preferably, the external guide sequencecomprises a ten to fifteen nucleotide sequence complementary to Zkun11mRNA, and a 3′-NCCA nucleotide sequence, wherein N is preferably apurine. The external guide sequence transcripts bind to the targetedmRNA species by the formation of base pairs between the mRNA and thecomplementary external guide sequences, thus promoting cleavage of mRNAby RNase P at the nucleotide located at the 5′-side of the base-pairedregion.

In general, the dosage of a composition comprising a therapeutic vectorhaving a Zkun11 nucleotide acid sequence, such as a recombinant virus,will vary depending upon such factors as the subject's age, weight,height, sex, general medical condition and previous medical history.Suitable routes of administration of therapeutic vectors includeintravenous injection, intraarterial injection, intraperitonealinjection, intramuscular injection, intratumoral injection, andinjection into a cavity that contains a tumor.

A composition comprising viral vectors, non-viral vectors, or acombination of viral and non-viral vectors of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby vectors or viruses are combined in amixture with a pharmaceutically acceptable carrier. As noted above, acomposition, such as phosphate-buffered saline is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient subject. Other suitable carriers are well-knownto those in the art (see, for example, Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985)).

For purposes of therapy, a therapeutic gene expression vector, or arecombinant virus comprising such a vector, and a pharmaceuticallyacceptable carrier are administered to a subject in a therapeuticallyeffective amount. A combination of an expression vector (or virus) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient subject.

When the subject treated with a therapeutic gene expression vector or arecombinant virus is a human, then the therapy is preferably somaticcell gene therapy. That is, the preferred treatment of a human with atherapeutic gene expression vector or a recombinant virus does notentail introducing into cells a nucleic acid molecule that can form partof a human germ line and be passed onto successive generations (i.e.,human germ line gene therapy).

14. Production of Transgenic Mice

Transgenic mice can be engineered to over-express the Zkun11 gene in alltissues or under the control of a tissue-specific or tissue-preferredregulatory element. These over-producers of Zkun11 can be used tocharacterize the phenotype that results from over-expression, and thetransgenic animals can serve as models for human disease caused byexcess Zkun11. Transgenic mice that over-express Zkun11 also providemodel bioreactors for production of Zkun11 in the milk or blood oflarger animals. Methods for producing transgenic mice are well-known tothose of skill in the art (see, for example, Jacob, “Expression andKnockout of Interferons in Transgenic Mice,” in Overexpression andKnockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124(Academic Press, Ltd. 1994), Monastersky and Robl (eds.), Strategies inTransgenic Animal Science (ASM Press 1995), and Abbud and Nilson,“Recombinant Protein Expression in Transgenic Mice,” in Gene ExpressionSystems: Using Nature for the Art of Expression, Fernandez and Hoeffler(eds.), pages 367-397 (Academic Press, Inc. 1999)).

For example, a method for producing a transgenic mouse that expresses aZkun11 gene can begin with adult, fertile males (studs) (B6C3f1, 2-8months of age (Taconic Farms, Germantown, N.Y.)), vasectomized males(duds) (B6D2f1, 2-8 months, (Taconic Farms)), prepubescent fertilefemales (donors) (B6C3f1, 4-5 weeks, (Taconic Farms)) and adult fertilefemales (recipients) (B6D2f1, 2-4 months, (Taconic Farms)). The donorsare acclimated for one week and then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company;St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfirmed by the presence of a vaginal plug the morning followingmating.

Fertilized eggs are collected under a surgical scope. The oviducts arecollected and eggs are released into urinanalysis slides containinghyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (described, for example, by Menino andO'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote4:129 (1996)) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are then stored in a 37° C./5% CO₂ incubator untilmicroinjection.

Ten to twenty micrograms of plasmid DNA containing a Zkun11 encodingsequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl(pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10nanograms per microliter for microinjection. For example, the Zkun11encoding sequences can encode the amino acid residues of SEQ ID NO:2.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pre-gassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, two-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans allowed to slide in. The peritoneal wall is closed with onesuture and the skin closed with a wound clip. The mice recuperate on a37° C. slide warmer for a minimum of four hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using, for example, a QIAGENDNEASY kit following the manufacturer's instructions. Genomic DNA isanalyzed by PCR using primers designed to amplify a Zkun11 gene or aselectable marker gene that was introduced in the same plasmid. Afteranimals are confirmed to be transgenic, they are back-crossed into aninbred strain by placing a transgenic female with a wild-type male, or atransgenic male with one or two wild-type female(s). As pups are bornand weaned, the sexes are separated, and their tails snipped forgenotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the zyphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum and the left lateral lobe ofthe liver exteriorized. Using 4-0 silk, a tie is made around the lowerlobe securing it outside the body cavity. An atraumatic clamp is used tohold the tie while a second loop of absorbable Dexon (American Cyanamid;Wayne, N.J.) is placed proximal to the first tie. A distal cut is madefrom the Dexon tie and approximately 100 mg of the excised liver tissueis placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of Zkun11 mRNA is examined for eachtransgenic mouse using an RNA solution hybridization assay or polymerasechain reaction.

In addition to producing transgenic mice that over-express Zkun11, it isuseful to engineer transgenic mice with either abnormally low or noexpression of the gene. Such transgenic mice provide useful models fordiseases associated with a lack of Zkun11. As discussed above, Zkun11gene expression can be inhibited using anti-sense genes, ribozyme genes,or external guide sequence genes. To produce transgenic mice thatunder-express the Zkun11 gene, such inhibitory sequences are targeted toZkun11 mRNA. Methods for producing transgenic mice that have abnormallylow expression of a particular gene are known to those in the art (see,for example, Wu et al., “Gene Underexpression in Cultured Cells andAnimals by Antisense DNA and RNA Strategies,” in Methods in GeneBiotechnology, pages 205-224 (CRC Press 1997)).

An alternative approach to producing transgenic mice that have little orno Zkun11 gene expression is to generate mice having at least one normalZkun11 allele replaced by a nonfunctional Zkun11 gene. One method ofdesigning a nonfunctional Zkun11 gene is to insert another gene, such asa selectable marker gene, within a nucleic acid molecule that encodesZkun11. Standard methods for producing these so-called “knockout mice”are known to those skilled in the art (see, for example, Jacob,“Expression and Knockout of Interferons in Transgenic Mice,” inOverexpression and Knockout of Cytokines in Transgenic Mice, Jacob(ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al., “NewStrategies for Gene Knockout,” in Methods in Gene Biotechnology, pages339-365 (CRC Press 1997)).

The present invention, thus generally described, will be understood morereadily by reference to the following example, which is provided by wayof illustration and is not intended to be limiting of the presentinvention.

EXAMPLE 1 Expression of the Zkun11 Gene

A panel of cDNA molecules from human tissues was screened for Zkun11expression using PCR. The panel included 78 marathon cDNA and cDNAsamples from various normal and cancerous human tissues and cell lines.The marathon cDNA molecules were produced using the MARATHON-READY kit(CLONTECH Laboratories, Inc.; Palo Alto, Calif.) and quality controltested with clathrin primers ZC21195 (5′ GAGGAGACCATAACCCCCGACAG 3′; SEQID NO:6) and ZC21196 (5′ CATAGCTCCCACCACACGATTTT 3′; SEQ ID NO:7), andthen diluted based on the intensity of the clathrin band.

To assure quality of the panel samples, three tests for quality controlwere run. First, to assess the RNA quality used for the libraries, thecDNA molecules were tested for average insert size by PCR with vectoroligonucleotides that were specific for the vector sequences for anindividual cDNA library. Second, standardization of the concentration ofthe cDNA in panel samples was achieved using standard PCR methods toamplify full length alpha tubulin or G3PDH cDNA using a 5′ vectoroligonucleotide ZC14063 (5′ CACCAGACATAATAGCTGACAGACT 3′; SEQ ID NO:8)and 3′ alpha tubulin specific oligo primer ZC17574 (5′GGTRTTGCTCAGCATGCACAC 3′; SEQ ID NO:9) or 3′ G3PDH specificoligonucleotide primer ZC17600 (5′ CATGTAGGCCATGAGGTCCACCAC 3′; SEQ IDNO:10). Third, a sample was sequenced to check for possible ribosomal ormitochondrial DNA contamination.

The panel was set up in a 96-well format that included a human genomicDNA (CLONTECH; Palo Alto, Calif.) positive control sample. Each wellcontained approximately 0.2-100 pg/μl of cDNA. To amplify Zkun11sequences, the PCR reactions were set up using oligonucleotides ZC37136(5′ CCTCTGAAGAAGGGCACCTGTAAT 3′; SEQ ID NO:11) and ZC37138 (5′GCCTCCACAGCCACTGAAGACA 3′; SEQ ID NO:12), TAKARA EX TAQ (Takara ShuzoCo. Ltd.; Nagoya, Japan), and Rediload dye (Research Gene tics, Inc.;Huntsville, Ala.). The amplification was carried out as follows: 1 cycleat 94° C. for 2 minutes, 35 cycles of 94° C. for 30 seconds, 63.1° C.for 30 seconds and 72° C. for 30 seconds, followed by 1 cycle at 72° C.for 5 minutes. About 10 μl of the PCR reaction product was fractionatedusing standard agarose gel electrophoresis in a 4% agarose gel. Thepredicted DNA fragment size of about 99 base pairs was observed ingenomic and prostate samples. In contrast, expression products were notdetected using the following tissues: adrenal gland, bladder, bonemarrow, cervix, colon, kidney, liver, lung, lymph node, mammary gland,ovary, pituitary, placenta, rectum, spleen, stomach, and testis.

The DNA fragments from genomic and prostate samples were excised andpurified using a Gel Extraction Kit (QIAGEN; Chatsworth, Calif.)according to manufacturer's instructions. Fragments were confirmed bysequencing to show that they were indeed Zkun11 sequences.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 12 <210> SEQ ID NO 1 <211> LENGTH: 153<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(153) <400> SEQUENCE: 1tgc aac tat cct ctg aag aag ggc acc tgt aa#t tca tat ttg acc aga       48Cys Asn Tyr Pro Leu Lys Lys Gly Thr Cys As #n Ser Tyr Leu Thr Arg 1               5   #                 10  #                 15ttc tac tac aac act ttg aca ttt tta tgt ga#g ccc ttt gtc ttc agt       96Phe Tyr Tyr Asn Thr Leu Thr Phe Leu Cys Gl #u Pro Phe Val Phe Ser             20      #             25      #             30ggc tgt gga ggc aac aga aac aac ttt aaa ca#g aaa tat ttc tgt gaa      144Gly Cys Gly Gly Asn Arg Asn Asn Phe Lys Gl #n Lys Tyr Phe Cys Glu         35          #         40          #         45aaa atg tgt               #                   #                  #        153 Lys Met Cys      50 <210> SEQ ID NO 2 <211> LENGTH: 51<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2Cys Asn Tyr Pro Leu Lys Lys Gly Thr Cys As #n Ser Tyr Leu Thr Arg 1               5   #                10   #                15Phe Tyr Tyr Asn Thr Leu Thr Phe Leu Cys Gl #u Pro Phe Val Phe Ser            20       #            25       #            30Gly Cys Gly Gly Asn Arg Asn Asn Phe Lys Gl #n Lys Tyr Phe Cys Glu        35           #        40           #        45 Lys Met Cys    50 <210> SEQ ID NO 3 <211> LENGTH: 153 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: This degenerate nucleotide #sequence encodes the       amino acid sequence of SEQ ID  #NO:2.<221> NAME/KEY: variation <222> LOCATION: (1)...(153)<223> OTHER INFORMATION: N is any nucleotide. <400> SEQUENCE: 3tgyaaytayc cnytnaaraa rggnacntgy aaywsntayy tnacnmgntt yt#aytayaay     60acnytnacnt tyytntgyga rccnttygtn ttywsnggnt gyggnggnaa ym#gnaayaay    120 ttyaarcara artayttytg ygaraaratg tgy       #                   #        153 <210> SEQ ID NO 4 <211> LENGTH: 16<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Peptide linker. <400> SEQUENCE: 4Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Se #r Gly Gly Gly Gly Ser 1               5   #                10   #                15<210> SEQ ID NO 5 <211> LENGTH: 51 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: motif. <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(51)<223> OTHER INFORMATION: Xaa is any amino acid. <400> SEQUENCE: 5Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xa #a Xaa Xaa Xaa Xaa Xaa 1               5   #                10   #                15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xa #a Xaa Xaa Xaa Xaa Xaa            20       #            25       #            30Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa #a Xaa Xaa Xaa Cys Xaa        35           #        40           #        45 Xaa Xaa Cys    50 <210> SEQ ID NO 6 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer. <400> SEQUENCE: 6gaggagacca taacccccga cag            #                  #                23 <210> SEQ ID NO 7 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer. <400> SEQUENCE: 7catagctccc accacacgat ttt            #                  #                23 <210> SEQ ID NO 8 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer. <400> SEQUENCE: 8caccagacat aatagctgac agact           #                  #               25 <210> SEQ ID NO 9 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer. <400> SEQUENCE: 9ggtrttgctc agcatgcaca c            #                  #                   #21 <210> SEQ ID NO 10 <211> LENGTH: 24<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer. <400> SEQUENCE: 10catgtaggcc atgaggtcca ccac           #                  #                24 <210> SEQ ID NO 11 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer. <400> SEQUENCE: 11cctctgaaga agggcacctg taat           #                  #                24 <210> SEQ ID NO 12 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer. <400> SEQUENCE: 12gcctccacag ccactgaaga ca            #                  #                 22

I claim:
 1. An isolated nucleic acid molecule that encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:2.
 2. The isolatednucleic acid molecule of claim 1, wherein the nucleic acid moleculeencodes a polypeptide consisting of the amino acid sequence of SEQ IDNO:2.
 3. The isolated nucleic acid molecule of claim 2, wherein thenucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:1.4. A vector, comprising the nucleic acid molecule of claim
 1. 5. Avector, comprising the nucleic acid molecule of claim
 3. 6. Arecombinant host cell, comprising an expression vector, wherein theexpression vector comprises: a nucleic acid molecule that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:2, atranscription promoter, and a transcription terminator, wherein thepromoter is operably linked to the nucleic acid molecule, and thenucleic acid molecule is operably linked to the transcriptionterminator, wherein the host cell is selected from the group consistingof bacterium, yeast cell, fungal cell, insect cell, avian cell,mammalian cell, and plant cell.
 7. The recombinant host cell of claim 6,wherein the host cell is a bacterium.
 8. The recombinant host cell ofclaim 6, the host cell is a yeast cell.
 9. The recombinant host cell ofclaim 6, the host cell is a fungal cell.
 10. The recombinant host cellof claim 6, wherein the host cell is an insect cell.
 11. The recombinanthost cell of claim 6, the host cell is an avian cell.
 12. Therecombinant host cell of claim 6, the host cell is a mammalian cell. 13.The recombinant host cell of claim 6, wherein the host cell is a plantcell.
 14. An expression vector, comprising a nucleic acid molecule thatencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2,a transcription promoter, and a transcription terminator, wherein thepromoter is operably linked to the nucleic acid molecule, and whereinthe nucleic acid molecule is operably linked to the transcriptionterminator.
 15. A method of producing a polypeptide that comprises theamino acid sequence of SEQ ID NO:2, the method comprising the step ofculturing recombinant host cells that comprise the expression vector ofclaim 14, and that produce the polypeptide.
 16. The method of claim 15,further comprising the step of isolating the polypeptide from thecultured recombinant host cells.